T cell receptor-like antibodies specific for a WTI peptide presented by HLA-A2

ABSTRACT

The present invention provides antigen binding proteins that specifically bind to Wilms&#39; tumor protein (WT1), including humanized, chimeric and fully human antibodies against WT1, antibody fragments, chimeric antigen receptors (CARs), fusion proteins, and conjugates thereof. The antigen binding proteins and antibodies bind to HLA-A0201-restricted WT1 peptide. Such antibodies, fragments, fusion proteins and conjugates thereof are useful for the treatment of WT1 associated cancers, including for example, breast cancer, ovarian cancer, prostate cancer, chronic myelocytic leukemia, multiple myeloma, acute lymphoblastic leukemia (ALL), acute myeloid/myelogenous leukemia (AML) and myelodysplastic syndrome (MDS). In more particular embodiments, the anti-WT1/A antibodies may comprise one or more framework region amino acid substitutions designed to improve protein stability, antibody binding and/or expression levels.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Patent application Ser. No.15/364,953, filed Nov. 30, 2016, which is a continuation of U.S. Patentapplication Ser. No. 14/724,155, filed May 28, 2015, which is acontinuation of U.S. Patent application Ser. No. 14/008,447, filed Dec.10, 2013, which is a National Phase filing under 35 U.S.C. § 371 of PCTInternational Application No. PCT/US2012/31892, filed Apr. 2, 2012,which claims the benefit of and priority to U.S. Provisional ApplicationNo. 61/470,635, filed Apr. 1, 2011, and U.S. Provisional Application No.61/491,392 filed May 31, 2011, all of which are hereby incorporated byreference in their entireties.

STATEMENT OF RIGHTS UNDER FEDERALLY-SPONSORED RESEARCH

This invention was made with government support under grants CA023766and CA055349 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

SEQUENCE LISTING

This application contains a Sequence Listing, created on Nov. 13, 2018;the file, in ASCII format, is named 115872-0392_Seqlisting.txt, and is80 KB in size. The file is hereby incorporated by reference in itsentirety into the application.

TECHNICAL FIELD

The present invention relates generally to antibodies against cytosolicproteins. More particularly, the invention relates to antibodies againstWilm's tumor oncogene protein (WT1), specifically antibodies thatrecognize a WT1 peptide in conjunction with a major histocompatabilityantigen.

BACKGROUND OF THE INVENTION

The Wilms' tumor oncogene protein (WT1) is an attractive target forimmunotherapy for most leukemias and a wide range of cancers. WT1 is azinc finger transcription factor that is normally expressed inmesodermal tissues during embryogenesis. In normal adult tissue, WT1expression is limited to low levels in CD34⁺ hematopoietic stem cellsbut is over-expressed in leukemias of multiple lineages and a wide rangeof solid tumors (1-2). More recently, WT1 expression has been reportedto be a marker of minimal residual disease. Increasing transcript levelsin patients with acute myeloid leukemia (AML) in morphologic remissionhave been predictive of overt clinical relapse (3, 4). Furthermore,antibodies to WT1 are detected in patients with hematopoieticmalignancies and solid tumors, indicating that WT1 is a highlyimmunogenic antigen (7).

For the most part, clinically approved therapeutic monoclonal antibodies(mAbs) recognize structures of cell surface proteins. WT1, however, is anuclear protein and, therefore, is inaccessible to classical antibodytherapy. Up until now, immunotherapy targeting WT1 has been limited tocellular approaches, exclusively aimed at generating WT1-specificcytotoxic CD8 T cell (CTL) responses that recognize peptides presentedon the cell surface by MHC class I molecules.

For induction of CTL responses, intracellular proteins are usuallydegraded by the proteasome or endo/lysosomes, and the resulting peptidefragments bind to MHC class I or II molecules. These peptide-MHCcomplexes are displayed at the cell surface where they provide targetsfor T cell recognition via a peptide-MHC (pMHC)-T cell receptor (TCR)interaction (8, 9). Vaccinations with peptides derived from the WT1protein induce HLA-restricted cytotoxic CD8 T cells, which are capableof killing tumor cells.

To improve efficacy, cancer antigens can be targeted with monoclonalantibody therapy. Monoclonal antibody (mAb) therapy has been shown toexert powerful antitumor effects by multiple mechanisms, includingcomplement-dependent cytotoxicity (CDC), antibody-dependent cellularcytotoxicity (ADCC) and direct cell inhibition or apoptosis-inducingeffects on tumor cells that over-express the target molecules.Furthermore, mAb can be used as carriers to specifically deliver acytotoxic moiety such as a radionuclide, cytotoxic drug or toxin to thetumor cells (18).

A tremendous benefit would exist if, in addition to a cellularimmunotherapy approach, a humoral immunotherapy approach was availableto target non-cell surface tumor antigens. Therefore, a monoclonalantibody (mAb) that mimics a T cell receptor in that it is specific fora target comprising a fragment of an intracellular protein inconjunction with an MHC molecule, for example, a WT1 peptide/HLA-A2complex, would be a novel and effective therapeutic agent alone or as avehicle capable of delivering potent anti-cancer reagents, such asdrugs, toxins and radioactive elements. Such mAbs would also be usefulas diagnostic or prognostic tools.

SUMMARY OF THE INVENTION

The present disclosure identifies and characterizes antigen-bindingproteins, such as antibodies, that are able to targetcytosolic/intracellular proteins, for example, the WT1 oncoprotein. Thedisclosed antibodies target a peptide/MHC complex as it would typicallyappear on the surface of a cell following antigen processing of WT1protein and presentation by the cell. In that regard, the antibodiesmimic T-cell receptors in that the antibodies have the ability tospecifically recognize and bind to a peptide in an MHC-restrictedfashion, that is, when the peptide is bound to an MHC antigen. Thepeptide/MHC complex recapitulates the antigen as it would typicallyappear on the surface of a cell following antigen processing andpresentation of the WT1 protein to a T-cell.

The antibodies disclosed specifically recognize and bind to epitopes ofa peptide/HLA-A2 complex, particularly a WT1/HLA-A0201 complex. Examplesof peptides that are recognized by the antigen-binding proteins of theinvention as part of an HLA-peptide complex include, but are not limitedto, those shown in Table 7, for example, a peptide with the amino acidsequence RMFPNAPYL (SEQ ID NO: 1.)

In one aspect, therefore, the invention relates to an isolated antibody,or antigen-binding fragment thereof, that binds to a peptide with theamino acid sequence, RMFPNAPYL, when said peptide is bound to an MHCantigen, such as HLA-A2.

In another aspect, the invention relates to an isolated antigen-bindingprotein, antibody, or antigen-binding fragment thereof, comprising (A)(i) a heavy chain (HC) variable region comprising HC-CDR1, HC-CDR2 andHC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 2, 3,and 4; and a light chain (LC) variable region comprising LC-CDR1,LC-CDR2 and LC-CDR3 respectively, comprising amino acid sequences SEQ IDNOS: 8, 9 and 10; (ii) a heavy chain (HC) variable region comprisingHC-CDR1, HC-CDR2 and HC-CDR3 respectively, comprising amino acidsequences SEQ ID NOS: 20, 21 and 22; and a light chain (LC) variableregion comprising LC-CDR1, LC-CDR2 and LC-CDR3 respectively, comprisingamino acid sequences SEQ ID NOS: 26, 27 and 28; (iii) a heavy chain (HC)variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3 respectively,comprising amino acid sequences SEQ ID NOS: 38, 39 and 40; and a lightchain (LC) variable region comprising LC-CDR1, LC-CDR2 and LC-CDR3respectively, comprising amino acid sequences selected from SEQ ID NOS:44, 45 and 46; (iv) a heavy chain (HC) variable region comprisingHC-CDR1, HC-CDR2 and HC-CDR3 respectively, comprising amino acidsequences SEQ ID NOS: 56, 57 and 58; and a light chain (LC) variableregion comprising LC-CDR1, LC-CDR2 and LC-CDR3 respectively, comprisingamino acid sequences SEQ ID NOS: 62, 63 and 64; (v) a heavy chain (HC)variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3 respectively,comprising amino acid sequences SEQ ID NOS: 74, 75 and 76; and a lightchain (LC) variable region comprising LC-CDR1, LC-CDR2 and LC-CDR3respectively, comprising amino acid sequences SEQ ID NOS: 80, 81 and 82;or (vi) a heavy chain (HC) variable region comprising HC-CDR1, HC-CDR2and HC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS:92, 93 and 94; and a light chain (LC) variable region comprisingLC-CDR1, LC-CDR2 and LC-CDR3 respectively, comprising amino acidsequences SEQ ID NOS: 98, 99 and 100.

In another aspect, the invention relates to an isolated antigen-bindingprotein, antibody, or antigen-binding fragment thereof, comprising aV_(H) and V_(L) comprising first and second amino acid sequences,respectively, selected from SEQ ID NOS: 14 and 16; 32 and 34; 50 and 52;68 and 70; 86 and 88; and 104 and 106.

In yet another aspect, the invention relates to an isolatedantigen-binding protein, antibody, or antigen-binding fragment thereof,comprising an amino acid sequence selected from SEQ ID NOS: 18, 36, 54,72, 90, and 108.

In a related aspect, the isolated antigen-binding protein comprises anantigen-binding region as disclosed in any of Tables 1-8. Theantigen-binding protein may be a fusion protein.

In another aspect, the invention relates to an immunoconjugatecomprising a first component which is an antigen-binding protein,antibody or antigen-binding fragment thereof as disclosed herein. Theimmunoconjugate comprises a second component that is a cytotoxin, adetectable label, a radioisotope, a therapeutic agent, a binding proteinor a molecule having a second amino acid sequence. Where the secondcomponent is a binding protein or second antibody, the binding proteinor second antibody has binding specificity for a target that isdifferent from the HLA-peptide complex for which the first is specific.

In a related aspect, therefore, the present invention relates tobispecific antibody comprising an antigen-binding protein or functionalfragment thereof as described herein.

In yet another aspect, the invention relates to nucleic acids thatencode antigen binding proteins, including antibodies and chimericantigen receptors specific for a WT1 peptide/HLA complex, in particularthe complex of WT1 peptide RMFPNAPYL/HLA-A0201.

In another related aspect, the invention relates to cells comprising thenucleic acids or antigen binding proteins disclosed herein, includingrecombinant immune effector cells, such as, T-cells genetically modifiedto express a chimeric antigen receptor comprising an antigen bindingregion in accordance with the present disclosure. Cells which have beenengineered to produce antibodies in accordance with the disclosure arealso encompassed by the invention.

In a related aspect, the invention relates to vectors comprising thenucleic acids to encode the antigen binding proteins disclosed herein,including vectors to facilitate expression and/or secretion of anantigen binding protein such as an antibody or chimeric antigen receptorin accordance with the present disclosure.

In a related aspect, the invention relates to pharmaceuticalcompositions comprising the antigen binding proteins, antibodies,nucleic acids, vectors or cells comprising the nucleic acids or antigenbinding proteins disclosed herein, together with a pharmaceuticallyacceptable carrier.

In another aspect, the invention relates to a method for detecting WT1on the surface of cells or tissues using WT1 antibodies of theinvention.

In yet another aspect, the invention relates to methods for treatment ofa subject having a WT1-positive disease, comprising administering to thesubject a therapeutically effective amount of an antigen bindingprotein, antibody or antigen binding fragment thereof, nucleic acidencoding the antigen binding protein or antibody or a cell comprisingthe nucleic acids or proteins as disclosed herein. The WT1-positivedisease is a chronic leukemia, acute leukemia or WT1⁺ cancer selectedfrom the group consisting of chronic myelocytic leukemia, multiplemyeloma (MM), acute lymphoblastic leukemia (ALL), acutemyeloid/myelogenous leukemia (AML), myelodysplastic syndrome (MDS),mesothelioma, ovarian cancer, gastrointestinal cancers, breast cancer,prostate cancer and glioblastoma. In some embodiments, the antigenbinding protein or antibody is a conjugate thereof having a cytotoxicmoiety linked thereto.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequence of Wilms tumor protein, (GenBankAccession No. P19544) with some HLA-restricted peptides bolded. The121-140 peptide further encompasses a 9-mer (underlined), RMFPNAPYL (SEQID NO: 1), which, in addition to analogs thereof, has been shown toinduce WT1-specific cytotoxic T-cell activity.

FIG. 2 is a graph showing that vaccination with WT1 peptides inducescytotoxic T cells against WT1⁺ leukemia cells.

FIG. 3 shows the results of a phage ELISA for specific binding of WT1/A2(WA) versus PBS control or R3/HLAA0201 (R3).

FIG. 4 shows specific binding of only WT1 phage antibodies that bind toT2 cells pulsed with WT1A peptide were selected.

FIG. 5 shows the binding affinity of a full-length IgG1 of a WT1antibody to RMF/A0201 complex tested by titration of the antibody atvarious concentrations. Results are shown for T2 cells pulsed with 50ug/ml RMF (upper panel). Control antibody is shown in the lower panel.

FIG. 6 shows the dependence on density of RMF/HLA-A0201 complexrecognized by WT1 antibody on T2 cells pulsed with RMF (upper panel) orcontrol, RHAMM-R3 (lower panel).

FIG. 7 shows an expression vector for expression of human antibodies.

FIG. 8 shows the results of SDS-PAGE analysis of WT1/A2 antibodies underreducing and non-reducing conditions.

FIG. 9 shows the results of kinetic binding analysis of an WT1/A2antibody demonstrating affinity of the antibody toward WT1/A2.

FIG. 10 shows the affinity (K_(D)) of antibody binding to WT1/A2complex.

FIG. 11 shows the mean fluorescence intensity (MFI) by flow cytometry ofpeptide titration on binding of some embodiments, mAb clone 5 (upperpanel), clone 15 (middle panel) and control (lower panel) to live T2cells pulsed with varying concentrations of peptide, WT1-A, WT1-A1 orcontrol.

FIG. 12 shows the results of peptide titration on binding of a WT1antibody, mAb 5 (upper panel), mAb 15 (lower panel) to live T2 cellspulsed with varying concentrations of WT1A peptide.

FIG. 13 shows the binding specificity of one embodiment, mAb 5, atdifferent concentrations (50 μg/ml upper; 25 μg/ml middle; and 12.5μg/ml lower) of peptide (R3, WT1-A1, WT1-A or no peptide.)

FIG. 14 shows the binding specificity of one embodiment, mAb 15, atdifferent concentrations (50 μg/ml upper; 25 μg/ml lower) of peptide(R3, WT1-A1, WT1-A or no peptide).

FIG. 15 shows dose-dependent binding of mAbs 5 (upper panel) and 15(lower panel) to T2 cells pulsed with WT1-A, WT1-A1, or RHAMM-R3peptide.

FIG. 16 shows binding of mAbs 5 and 15 to U266, a myeloma cell line.

FIG. 17 shows binding of mAb 15 to BV173, a cell line derived from anindividual with (Ph1)-positive acute leukemia.

FIG. 18 shows the specific binding of ESK1 (#13) to WT1/A2 complex onthe surface of T2 cells pulsed with WT1 peptide.

FIG. 19 and FIG. 20 show that WT1 antibody is able to recognize RMFpeptide in which substitution of different positions of the RMF peptidewith alanine is made (see also Table 10) and that the loss of bindingseen with substitution of position 1 by either alanine (WT1-A1-B) ortyrosine (WT1-A1), was not due to the reduction of peptide bindingaffinity to the HLA-A2 molecule, as both peptides showed the strongestbinding in T2 stabilization assay using the mAb specific for the HLA-A2molecule, clone BB7.

FIG. 21 shows recognition by WT1 antibody of naturally presentedRMF/HLA-A0201 complex on the cell surface of human mesothelioma celllines, JMN (WT1⁺/A0201⁺) but not MSTO (WT1⁺/HLA-A0201⁻).

FIG. 22 shows binding of WT1 antibodies to human CML-derived cell lineBV173.

FIG. 23 is a Scatchard analysis based on binding of WT1 antibody to JMNcells and shows an avidity constant of about 0.2 nM.

FIG. 24 shows WT1 antibody binding to a panel of mesothelioma andleukemia cells.

FIG. 25 shows the results of flow cytometric analyses gated on CD33 andCD34 double positive AML blast cells from an HLA-A2 positive patient.ESK1 binds to the leukemia blasts.

FIG. 26 shows the results of flow cytometric analyses gated on CD33 andCD34 double positive AML blast cells from an HLA-A2 negative patient.WT1 mAb ESK1 did not bind to the blasts.

FIG. 27 shows WT1 mAb ESK1 mediated ADCC against T2 cells pulsed withRMF peptide.

FIG. 28 shows the ability of WT1 antibody to mediate ADCC with humaneffectors in JMN and leukemia cell line BV173 (lower panel) but not MSTOcells.

FIG. 29 shows that WT1 mAbs are effective against human leukemia cellline BV173 but not HL60 cells, which are not HLA-A2⁺.

FIG. 30 shows that WT1 antibody induces ADCC against primary AML blastsfrom an HLA-A2 positive patient.

FIG. 31 shows the results of treatment of human BV173 in NSG mice usingantibodies of the invention.

FIG. 32 shows that at later time points, mice treated with WT1 antibodyonly began to relapse, while antibody with effectors cured 2 of 5 mice.

FIG. 33 shows that WT1 antibody significantly reduces tumor burden in adose-dependent manner.

FIG. 34 shows that antibody with altered carbohydrate in Fc (MAGE) ismore active in ADCC than original antibody.

DETAILED DESCRIPTION OF THE INVENTION

All publications, patents and other references cited herein areincorporated by reference in their entirety into the present disclosure.

In practicing the present invention, many conventional techniques inmolecular biology, microbiology, cell biology, biochemistry, andimmunology are used, which are within the skill of the art. Thesetechniques are described in greater detail in, for example, MolecularCloning: a Laboratory Manual 3rd edition, J. F. Sambrook and D. W.Russell, ed. Cold Spring Harbor Laboratory Press 2001; RecombinantAntibodies for Immunotherapy, Melvyn Little, ed. Cambridge UniversityPress 2009; “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “AnimalCell Culture” (R. I. Freshney, ed., 1987); “Methods in Enzymology”(Academic Press, Inc.); “Current Protocols in Molecular Biology” (F. M.Ausubel et al., eds., 1987, and periodic updates); “PCR: The PolymeraseChain Reaction”, (Mullis et al., ed., 1994); “A Practical Guide toMolecular Cloning” (Perbal Bernard V., 1988); “Phage Display: ALaboratory Manual” (Barbas et al., 2001). The contents of thesereferences and other references containing standard protocols, widelyknown to and relied upon by those of skill in the art, includingmanufacturers' instructions are hereby incorporated by reference as partof the present disclosure. The following abbreviations are usedthroughout the application:

Ab: Antibody

ADCC: Antibody-dependent cellular cytotoxicity

ALL: Acute lymphocytic leukemia

AML: Acute myeloid leukemia

APC: Antigen presenting cell

β2M: Beta-2-microglobulin

BiTE: Bi-specific T cell engaging antibody

CAR: Chimeric antigen receptor

CDC: Complement dependent cytotoxicity

CMC: Complement mediated cytotoxicity

CDR: Complementarity determining region (see also HVR below)

C_(L): Constant domain of the light chain

CH₁: 1^(st) constant domain of the heavy chain

CH_(1,2,3): 1^(st), 2^(nd) and 3^(rd) constant domains of the heavychain

CH_(2,3): 2^(nd) and 3^(rd) constant domains of the heavy chain

CHO: Chinese hamster ovary

CTL: Cytotoxic T cell

E:T Ratio: Effector:Target ratio

Fab: Antibody binding fragment

FACS: Flow assisted cytometric cell sorting

FBS: Fetal bovine serum

FR: Framework region

HC: Heavy chain

HLA: Human leukocyte antigen

HVR-H: Hypervariable region-heavy chain (see also CDR)

HVR-L: Hypervariable region-light chain (see also CDR)

Ig: Immunoglobulin

IRES: Internal ribosome entry site

K_(D): Dissociation constant

k_(off): Dissociation rate

k_(on): Association rate

MHC: Major histocompatibility complex

MM: Multiple myeloma

scFv: Single-chain variable fragment

TCR: T cell receptor

V_(H): Variable heavy chain includes heavy chain hypervariable regionand heavy chain variable framework region

V_(L): Variable light chain includes light chain hypervariable regionand light chain variable framework region

WT1: Wilms tumor protein 1

In the description that follows, certain conventions will be followed asregards the usage of terminology. Generally, terms used herein areintended to be interpreted consistently with the meaning of those termsas they are known to those of skill in the art.

An “antigen-binding protein” is a protein or polypeptide that comprisesan antigen-binding region or antigen-binding portion, that is, has astrong affinity to another molecule to which it binds. Antigen-bindingproteins encompass antibodies, chimeric antigen receptors (CARs) andfusion proteins.

“Antibody” and “antibodies” as those terms are known in the art refer toantigen binding proteins of the immune system. The term “antibody” asreferred to herein includes whole, full length antibodies having anantigen-binding region, and any fragment thereof in which the“antigen-binding portion” or “antigen-binding region” is retained, orsingle chains, for example, single chain variable fragment (scFv),thereof. A naturally occurring “antibody” is a glycoprotein comprisingat least two heavy (H) chains and two light (L) chains inter-connectedby disulfide bonds. Each heavy chain is comprised of a heavy chainvariable region (abbreviated herein as V_(H)) and a heavy chain constant(CH) region. The heavy chain constant region is comprised of threedomains, CH1, CH2 and CH3. Each light chain is comprised of a lightchain variable region (abbreviated herein as V_(L)) and a light chainconstant C_(L) region. The light chain constant region is comprised ofone domain, C_(L). The V_(H) and V_(L) regions can be further subdividedinto regions of hypervariability, termed complementarity determiningregions (CDR), interspersed with regions that are more conserved, termedframework regions (FR). Each V_(H) and V_(L) is composed of three CDRsand four FRs arranged from amino-terminus to carboxy-terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variableregions of the heavy and light chains contain a binding domain thatinteracts with an antigen. The constant regions of the antibodies maymediate the binding of the immunoglobulin to host tissues or factors,including various cells of the immune system (e.g., effector cells) andthe first component (C1q) of the classical complement system.

The term “antigen-binding portion” or “antigen-binding region” of anantibody, as used herein, refers to that region or portion of theantibody that binds to the antigen and which confers antigen specificityto the antibody; fragments of antigen-binding proteins, for example,antibodies includes one or more fragments of an antibody that retain theability to specifically bind to an antigen (e.g., an peptide/HLAcomplex). It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody.Examples of antigen-binding fragments encompassed within the term“antibody fragments” of an antibody include a Fab fragment, a monovalentfragment consisting of the V_(L), V_(H), C_(L) and CH1 domains; a F(ab)₂fragment, a bivalent fragment comprising two Fab fragments linked by adisulfide bridge at the hinge region; a Fd fragment consisting of theV_(H) and CH1 domains; a Fv fragment consisting of the V_(L) and V_(H)domains of a single arm of an antibody; a dAb fragment (Ward et al.,1989 Nature 341:544-546), which consists of a V_(H) domain; and anisolated complementarity determining region (CDR).

Furthermore, although the two domains of the Fv fragment, V_(L) andV_(H), are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the V_(L) and V_(H) regions pair toform monovalent molecules. These are known as single chain Fv (scFv);see e.g., Bird et al., 1988 Science 242:423-426; and Huston et al., 1988Proc. Natl. Acad. Sci. 85:5879-5883. These antibody fragments areobtained using conventional techniques known to those of skill in theart, and the fragments are screened for utility in the same manner asare intact antibodies.

An “isolated antibody” or “isolated antigen-binding protein” is onewhich has been identified and separated and/or recovered from acomponent of its natural environment. “Synthetic antibodies” or“recombinant antibodies” are generally generated using recombinanttechnology or using peptide synthetic techniques known to those of skillin the art.

Traditionally, the MHC-peptide complex could only be recognized by aT-cell receptor (TCR), limiting our ability to detect an epitope ofinterest using T cell-based readout assays. In the present disclosure,antigen binding proteins, including antibodies, having anantigen-binding region based on scFvs that are selected from human scFvphage display libraries using recombinant HLA-peptide complexes aredescribed. These molecules demonstrated exquisite specificity, forexample as shown with anti-WT1 antibodies that recognize onlyHLA-A2-RMFPNAPYL complexes. In addition, along with their inability tobind to HLA-complexes containing other peptides, the molecules were alsounable to bind to the peptides themselves, further demonstrating theirTCR-like specificity.

The scFvs of the disclosure selected by phage display were initiallytested for their ability to bind to peptide presented on the surface ofHLA-positive cells. After T2 cells were incubated in the presence ofpeptide, fluorescently labeled antibodies could be used to selectivelyrecognize the antigen pulsed cells using flow cytometry.

In some embodiments, the invention includes antibodies that have thescFv sequence fused to one or more constant domains of the heavy to forman antibody with an Fc region of a human immunoglobulin to yield abivalent protein, increasing the overall avidity and stability of theantibody. In addition, the Fc portion allows the direct conjugation ofother molecules, including but not limited to fluorescent dyes,cytotoxins, radioisotopes etc. to the antibody for example, for use inantigen quantitation studies, to immobilize the antibody for affinitymeasurements, for targeted delivery of a therapeutic agent, to test forFc-mediated cytotoxicity using immune effector cells and many otherapplications.

The results presented here highlight the specificity, sensitivity andutility of the antibodies of the invention in targeting MHC-peptidecomplexes.

The molecules of the invention are based on the identification andselection of single chain variable fragments (scFv) using phage display,the amino acid sequence of which confers the molecules' specificity forthe MHC restricted peptide of interest and forms the basis of allantigen binding proteins of the disclosure. The scFv, therefore, can beused to design a diverse array of “antibody” molecules, including, forexample, full length antibodies, fragments thereof, such as Fab andF(ab′)₂, minibodies, fusion proteins, including scFv-Fc fusions,multivalent antibodies, that is, antibodies that have more than onespecificity for the same antigen or different antigens, for example,bispecific T-cell engaging antibodies (BiTe), tribodies, etc. (seeCuesta et al., Multivalent antibodies: when design surpasses evolution.Trends in Biotechnology 28:355-362 2010).

In an embodiment in which the antigen-binding protein is a full lengthantibody, the heavy and light chains of an antibody of the invention maybe full-length (e.g., an antibody can include at least one, andpreferably two, complete heavy chains, and at least one, and preferablytwo, complete light chains) or may include an antigen-binding portion (aFab, F(ab′)₂, Fv or a single chain Fv fragment (“scFv”)). In otherembodiments, the antibody heavy chain constant region is chosen from,e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE. In someembodiments, the immunoglobulin isotype is selected from IgG1, IgG2,IgG3, and IgG4, more particularly, IgG1 (e.g., human IgG1). The choiceof antibody type will depend on the immune effector function that theantibody is designed to elicit.

In constructing a recombinant immunoglobulin, appropriate amino acidsequences for constant regions of various immunoglobulin isotypes andmethods for the production of a wide array of antibodies are known tothose of skill in the art.

In one embodiment, the antibody or other antigen binding protein is ananti-WT1/HLA-A2 scFv or antigen-binding fragment thereof having anantigen binding region that comprises the amino acid sequence of SEQ IDNO: 18 and specifically binds to a peptide with the amino acid sequenceRMFPNAPYL (SEQ ID NO: 1) in conjunction with HLA-A0201. In someembodiments, the anti-WT1 antibody is a scFv-Fc fusion protein or fulllength human IgG with VH and VL regions or CDRs selected from Table 1.

TABLE 1 Antigen WT1 (Ext002 #3) Peptide RMFPNAPYL (SEQ ID NO: 1) CDRs: 12 3 VH GGTFSSYAIS GIIPIFGTANYAQKFQG RIPPYYGMDV (SEQ ID NO: 2)(SEQ ID NO: 3) (SEQ ID NO: 4) DNA ggaggcaccttcagcaggggatcatccctatctttggtac cggattcccccgtactacggtat ctatgctatcagcagcaaactacgcacagaagtt ggacgtc (SEQ ID NO: 5) ccagggc (SEQ ID NO: 7)(SEQ ID NO: 6) VL SGSSSNIGSNYVY RSNQRPS AAWDDSLNGVV (SEQ ID NO: 8)(SEQ ID NO: 9) (SEQ ID NO: 10) DNA tctggaagcagctccaacaggagtaatcagcggccctca gcagcatgggatgacagcctg atcggaagtaattatgtat(SEQ ID NO: 12) aatggtgtggta ac (SEQ ID NO: 13) (SEQ ID NO: 11) FullQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLE VHWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARRIPPYYGMDVWGQGTTVTVSS (SEQ ID NO: 14) DNAcaggtgcagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaaggcttctggaggcaccttcagcagctatgctatcagctgggtgcgacaggcccctggacaagggcttgagtggatgggagggatcatccctatctttggtacagcaaactacgcacagaagttccagggcagagtcacgattaccgcggacgaatccacgagcacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcgagacggattcccccgtactacggtatggacgtctggggccaagggaccacggtcaccgtctcctca (SEQ ID NO: 15) FullQTVVTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKL VLLIYRSNQRPSGVPDRFSGSKSGTSASLAISGPRSVDEADYYCAAWDD SLNGVVFGGGTKLTVLG(SEQ ID NO: 16) DNAcagactgtggtgactcagccaccctcagcgtctgggacccccgggcagagggtcaccatctcttgttctggaagcagctccaacatcggaagtaattatgtatactggtaccaacagctcccaggaacggcccccaaactcctcatctataggagtaatcagcggccctcaggggtccctgaccgattctctggctccaagtctggcacctcagcctccctggccatcagtgggccccggtccgtggatgaggctgattattactgtgcagcatgggatgacagcctgaatggtgtggtattcggcggagggaccaagctgaccgtcctagg t(SEQ ID NO: 17) scFv QTVVTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRSNQRPSGVPDRFSGSKSGTSASLAISGPRSVDEADYYCAAWDDSLNGVVFGGGTKLTVLGSRGGGGSGGGGSGGGSLEMAQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARRIPPYY GMDVWGQGTTVTVSS(SEQ ID NO: 18) DNAcagactgtggtgactcagccaccctcagcgtctgggacccccgggcagagggtcaccatctcttgttctggaagcagctccaacatcggaagtaattatgtatactggtaccaacagctcccaggaacggcccccaaactcctcatctataggagtaatcagcggccctcaggggtccctgaccgattctctggctccaagtctggcacctcagcctccctggccatcagtgggccccggtccgtggatgaggctgattattactgtgcagcatgggatgacagcctgaatggtgtggtattcggcggagggaccaagctgaccgtcctaggttctagaggtggtggtggtagcggcggcggcggctctggtggtggatccctcgagatggcccaggtgcagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaaggcttctggaggcaccttcagcagctatgctatcagctgggtgcgacaggcccctggacaagggcttgagtggatgggagggatcatccctatctttggtacagcaaactacgcacagaagttccagggcagagtcacgattaccgcggacgaatccacgagcacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcgagacggattcccccgtactacggtatggacgtctggggccaagggaccacggtcaccgtctcctca (SEQ ID NO: 19)

In another embodiment, the antibody or antigen binding protein is ananti-WT1 scFv or antigen-binding fragment thereof that has an antigenbinding region that comprises the amino acid sequence of SEQ ID NO: 36and specifically binds to a peptide with the amino acid sequenceRMFPNAPYL (SEQ ID NO: 1) in conjunction with HLA-A0201. In otherembodiments, the anti-WT-1 antibody is a scFv-Fc fusion protein or fulllength human IgG with VH and VL regions or CDRs selected from Table 2.

TABLE 2 Antigen WT1 (Ext002 #5) Peptide RMFPNAPYL (SEQ ID NO: 1) CDRs: 12 3 VH GDSVSSNSAAWN RTYYGSKWYNDYAVS GRLGDAFDI (SEQ ID NO: 20) VKS(SEQ ID NO: 22) (SEQ ID NO: 21) DNA ggggacagtgtctctagcaggacatactacgggtccaag ggtcgcttaggggatgcttttga aacagtgctgcttggaactggtataatgattatgcagtatct tatc (SEQ ID NO: 23) gtgaaaagt (SEQ ID NO: 25)(SEQ ID NO: 24) VL RASQSISSYLN AASSLQS QQSYSTPLT (SEQ ID NO: 26)(SEQ ID NO: 27) (SEQ ID NO: 28) DNA cgggcaagtcagagcattgctgcatccagtttgcaaagt caacagagttacagtacccct agcagctatttaaat(SEQ ID NO: 30) ctcact (SEQ ID NO: 29) (SEQ ID NO: 31) FullQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGL VHEWLGRTYYGSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARGRLGDAFDIWGQGTMVTVSS (SEQ ID NO: 32) DNAcaggtacagctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctcactcacctgtgccatctccggggacagtgtctctagcaacagtgctgcttggaactggatcaggcagtccccatcgagaggccttgagtggctgggaaggacatactacgggtccaagtggtataatgattatgcagtatctgtgaaaagtcgaataaccatcaacccagacacatccaagaaccagttctccctgcagctgaactctgtgactcccgaggacacggctgtgtattactgtgcaagaggtcgcttaggggatgcttttgatatctggggccaagggacaatggtcaccgtctcttca (SEQ ID NO: 33) FullDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY VLAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLT FGGGTKVDIKR(SEQ ID NO: 34) DNAgacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaacagagttacagtacccctctcactttcggcggagggaccaaagtggatatcaaacgt (SEQ ID NO: 35)scFv DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVDIKRSRGGGGSGGGGSGGGGSLEMAQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYGSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARGRLGDAF DIWGQGTMVTVSS(SEQ ID NO: 36) DNAgacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaacagagttacagtacccctctcactttcggcggagggaccaaagtggatatcaaacgttctagaggtggtggtggtagcggcggcggcggctctggtggtggtggatccctcgagatggcccaggtacagctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctcactcacctgtgccatctccggggacagtgtctctagcaacagtgctgcttggaactggatcaggcagtccccatcgagaggccttgagtggctgggaaggacatactacgggtccaagtggtataatgattatgcagtatctgtgaaaagtcgaataaccatcaacccagacacatccaagaaccagttctccctgcagctgaactctgtgactcccgaggacacggctgtgtattactgtgcaagaggtcgcttaggggatgcttttgatatctggggccaagggacaatggtcaccgtctcttca (SEQ ID NO: 37)

In another embodiment, the antibody or antigen binding protein is ananti-WT1 scFv or antigen binding fragment thereof that has an antigenbinding region that comprises the amino acid sequence of SEQ ID NO: 54and specifically binds to a peptide with the amino acid sequenceRMFPNAPYL (SEQ ID NO: 1) in conjunction with HLA-A0201. In otherembodiments, the anti-WT-1 antibody is a scFv-Fc fusion protein or fulllength human IgG with VH and VL regions or CDRs selected from Table 3.

TABLE 3 Antigen WT1 (Ext002 #13) Peptide RMFPNAPYL (SEQ ID NO: 1) CDRs:1 2 3 VH GYSFTNFWIS RVDPGYSYSTYSPSF VQYSGYYDWFDP (SEQ ID NO: 38) QG(SEQ ID NO: 40) (SEQ ID NO: 39) DNA ggatacagcttcaccaactagggttgatcctggctactctta gtacaatatagtggctactatg tctggatcagctagcacctacagcccgtccttc actggttcgacccc (SEQ ID NO: 41) caaggc(SEQ ID NO: 43) (SEQ ID NO: 42) VL SGSSSNIGSNTVN SNNQRPS AAWDDSLNGWV(SEQ ID NO: 44) (SEQ ID NO: 45) (SEQ ID NO: 46) DNA tctggaagcagctccaacagtaataatcagcggccctca gcagcatgggatgacagcct atcggaagtaatactgtaa(SEQ ID NO: 48) gaatggttgggtg ac (SEQ ID NO: 49) (SEQ ID NO: 47) Full VHQMQLVQSGAEVKEPGESLRISCKGSGYSFTNFWISWVRQMPGKGLEWMGRVDPGYSYSTYSPSFQGHVTISADKSTSTAYLQWNSLKASDTAMYYCARVQYSGYYDWFDPWGQGTLVTVSS (SEQ ID NO: 50) DNAcagatgcagctggtgcagtccggagcagaggtgaaagagcccggggagtctctgaggatctcctgtaagggttctggatacagcttcaccaacttctggatcagctgggtgcgccagatgcccgggaaaggcctggagtggatggggagggttgatcctggctactcttatagcacctacagcccgtccttccaaggccacgtcaccatctcagctgacaagtctaccagcactgcctacctgcagtggaacagcctgaaggcctcggacaccgccatgtattactgtgcgagagtacaatatagtggctactatgactggttcgacccctggggccagggaaccctggtcaccgtctcctca (SEQ ID NO: 51) FullQAVVTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQVPGTAPK VLLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWD DSLNGWVFGGGTKLTVLG(SEQ ID NO: 52) DNAcaggctgtggtgactcagccaccctcagcgtctgggacccccgggcagagggtcaccatctcttgttctggaagcagctccaacatcggaagtaatactgtaaactggtaccagcaggtcccaggaacggcccccaaactcctcatctatagtaataatcagcggccctcaggggtccctgaccgattctctggctccaagtctggcacctcagcctccctggccatcagtgggctccagtctgaggatgaggctgattattactgtgcagcatgggatgacagcctgaatggttgggtgttcggcggagggaccaagctgaccgtcct aggt(SEQ ID NO: 53) scFv QAVVTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQVPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGWVFGGGTKLTVLGSRGGGGSGGGGSGGGGSLEMAQMQLVQSGAEVKEPGESLRISCKGSGYSFTNFWISWVRQMPGKGLEWMGRVDPGYSYSTYSPSFQGHVTISADKSTSTAYLQWNSLKASDTAMYYCA RVQYSGYYDWFDPWGQGTLVTVSS(SEQ ID NO: 54) DNAcaggctgtggtgactcagccaccctcagcgtctgggacccccgggcagagggtcaccatctcttgttctggaagcagctccaacatcggaagtaatactgtaaactggtaccagcaggtcccaggaacggcccccaaactcctcatctatagtaataatcagcggccctcaggggtccctgaccgattctctggctccaagtctggcacctcagcctccctggccatcagtgggctccagtctgaggatgaggctgattattactgtgcagcatgggatgacagcctgaatggttgggtgttcggcggagggaccaagctgaccgtcctaggttctagaggtggtggtggtagcggcggcggcggctctggtggtggtggatccctcgagatggcccagatgcagctggtgcagtccggagcagaggtgaaagagcccggggagtctctgaggatctcctgtaagggttctggatacagcttcaccaacttctggatcagctgggtgcgccagatgcccgggaaaggcctggagtggatggggagggttgatcctggctactcttatagcacctacagcccgtccttccaaggccacgtcaccatctcagctgacaagtctaccagcactgcctacctgcagtggaacagcctgaaggcctcggacaccgccatgtattactgtgcgagagtacaatatagtggctactatgactggttcgacccctggggccagggaaccctggtcaccgtctcctca (SEQ ID NO: 55)

In another embodiment, the antibody or antigen binding protein is ananti-WT1 scFv or antigen binding fragment thereof that has an antigenbinding region that comprises the amino acid sequence of SEQ ID NO: 72and specifically binds to a peptide with the amino acid sequenceRMFPNAPYL (SEQ ID NO:1) in conjunction with HLA-A0201. In otherembodiments, the anti-WT-1 antibody is a scFv-Fc fusion protein or fulllength human IgG with VH and VL regions or CDRs selected from Table 4.

TABLE 4 Antigen WT1 (Ext002 #15) Peptide RMFPNAPYL (SEQ ID NO: 1) CDRs:1 2 3 VH GYNFSNKWIG IIYPGYSDITYSPSFQG HTALAGFDY (SEQ ID NO: 56)(SEQ ID NO: 57) (SEQ ID NO: 58) DNA ggctacaactttagcaacaatcatctatcccggttactcgga cacacagctttggccggctttg agtggatcggccatcacctacagcccgtccttc actac (SEQ ID NO: 59) caaggc (SEQ ID NO: 61)(SEQ ID NO: 60) VL RASQNINKWLA KASSLES QQYNSYAT (SEQ ID NO: 62)(SEQ ID NO: 63) (SEQ ID NO: 64) DNA Cgggccagtcagaatatcaaggcgtctagtttagaaagt caacaatataatagttatgcga aataagtggctggcc(SEQ ID NO: 66) cg (SEQ ID NO: 65) (SEQ ID NO: 67) FullQVQLVQSGAEVKKPGESLKISCKGSGYNFSNKWIGWVRQLPGRGLE VHWIAIIYPGYSDITYSPSFQGRVTISADTSINTAYLHWHSLKASDTAMYYCVRHTALAGFDYWGLGTLVTVSS (SEQ ID NO: 68) DNAcaggtgcagctggtgcagtctggagcagaggtgaaaaagcccggagagtctctgaagatctcctgtaagggttctggctacaactttagcaacaagtggatcggctgggtgcgccaattgcccgggagaggcctggagtggatagcaatcatctatcccggttactcggacatcacctacagcccgtccttccaaggccgcgtcaccatctccgccgacacgtccattaacaccgcctacctgcactggcacagcctgaaggcctcggacaccgccatgtattattgtgtgcgacacacagctttggccggctttgactactggggcctgggcaccctggtcaccgtctcctca (SEQ ID NO: 69) FullDIQMTQSPSTLSASVGDRVTITCRASQNINKWLAWYQQRPGKAPQLLI VLYKASSLESGVPSRFSGSGSGTEYTLTISSLQPDDFATYYCQQYNSYAT FGQGTKVEIKR(SEQ ID NO: 70) DNAgacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcacaatcacttgccgggccagtcagaatatcaataagtggctggcctggtatcagcagagaccagggaaagcccctcagctcctgatctataaggcgtctagtttagaaagtggggtcccatctaggttcagcggcagtggatctgggacagaatacactctcaccatcagcagcctgcagcctgatgattttgcaacttattactgccaacaatataatagttatgcgacgttcggccaagggaccaaggtggaaatcaaacgt (SEQ ID NO: 71)scFv DIQMTQSPSTLSASVGDRVTITCRASQNINKWLAWYQQRPGKAPQLLIYKASSLESGVPSRFSGSGSGTEYTLTISSLQPDDFATYYCQQYNSYATFGQGTKVEIKRSRGGGGSGGGGSGGGGSLEMAQVQLVQSGAEVKKPGESLKISCKGSGYNFSNKWIGWVRQLPGRGLEWIAIIYPGYSDITYSPSFQGRVTISADTSINTAYLHWHSLKASDTAMYYCVRHTALAGFDYWGL GTLVTVSS(SEQ ID NO: 72) DNAgacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcacaatcacttgccgggccagtcagaatatcaataagtggctggcctggtatcagcagagaccagggaaagcccctcagctcctgatctataaggcgtctagtttagaaagtggggtcccatctaggttcagcggcagtggatctgggacagaatacactctcaccatcagcagcctgcagcctgatgattttgcaacttattactgccaacaatataatagttatgcgacgttcggccaagggaccaaggtggaaatcaaacgttctagaggtggtggtggtagcggcggcggcggctctggtggtggtggatccctcgagatggcccaggtgcagctggtgcagtctggagcagaggtgaaaaagcccggagagtctctgaagatctcctgtaagggttctggctacaactttagcaacaagtggatcggctgggtgcgccaattgcccgggagaggcctggagtggatagcaatcatctatcccggttactcggacatcacctacagcccgtccttccaaggccgcgtcaccatctccgccgacacgtccattaacaccgcctacctgcactggcacagcctgaaggcctcggacaccgccatgtattattgtgtgcgacacacagctttggccggctttgactactggggcctgggcaccctggtcaccgtctcctca (SEQ ID NO: 73)

In another embodiment, the antibody or antigen binding protein is ananti-WT1 scFv or antigen binding fragment thereof that has an antigenbinding region that comprises the amino acid sequence of SEQ ID NO: 90and specifically binds to a peptide with the amino acid sequenceRMFPNAPYL (SEQ ID NO: 1) in conjunction with HLA-A0201. In otherembodiments, the anti-WT-1 antibody is a scFv-Fc fusion protein or fulllength human IgG with VH and VL regions or CDRs selected from Table 5.

TABLE 5 Antigen WT1 (Ext002 #18) Peptide RMFPNAPYL (SEQ ID NO: 1) CDRs:1 2 3 VH GFTFDDYGMS GINWNGGSTGYADS ERGYGYHDPHDY (SEQ ID NO: 74) VRG(SEQ ID NO: 76) (SEQ ID NO: 75) DNA gggttcacctttgatgattatggtattaattggaatggtggt gagcgtggctacgggtacca ggcatgagcagcacaggttatgcagactc tgatccccatgactac (SEQ ID NO: 77) tgtgaggggc(SEQ ID NO: 79) (SEQ ID NO: 78) VL GRNNIGSKSVH DDSDRPS QVWDSSSDHVV(SEQ ID NO: 80) (SEQ ID NO: 81) (SEQ ID NO: 82) DNA gggagaaacaacattgggatgatagcgaccggccctc caggtgtgggatagtagtagt aagtaaaagtgtgcac agatcatgtggta (SEQ ID NO: 83) (SEQ ID NO: 84) (SEQ ID NO: 85) FullEVQLVQSGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKG VHLEWVSGINWNGGSTGYADSVRGRFTISRDNAKNSLYLQMNSLRAEDTALYYCARERGYGYHDPHDYWGQGTLVTVSS (SEQ ID NO: 86) DNAgaagtgcagctggtgcagtctgggggaggtgtggtacggcctggggggtccctgagactctcctgtgcagcctctgggttcacctttgatgattatggcatgagctgggtccgccaagctccagggaaggggctggagtgggtctctggtattaattggaatggtggtagcacaggttatgcagactctgtgaggggccgattcaccatctccagagacaacgccaagaactccctgtatctgcaaatgaacagtctgagagccgaggacacggccttgtattactgtgcgagagagcgtggctacgggtaccatgatccccatgactactggggccaaggcaccctggtgaccgtctcctca (SEQ ID NO: 87) FullQSVVTQPPSVSVAPGKTARITCGRNNIGSKSVHWYQQKPGQAPVL VLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVW DSSSDHVVFGGGTKLTVLG(SEQ ID NO: 88) DNAcagtctgtcgtgacgcagccgccctcggtgtcagtggccccaggaaagacggccaggattacctgtgggagaaacaacattggaagtaaaagtgtgcactggtaccagcagaagccaggccaggcccctgtgctggtcgtctatgatgatagcgaccggccctcagggatccctgagcgattctctggctccaactctgggaacacggccaccctgaccatcagcagggtcgaagccggggatgaggccgactattactgtcaggtgtgggatagtagtagtgatcatgtggtattcggcggagggaccaagctgaccgtcctaggt (SEQ ID NO: 89) scFvQSVVTQPPSVSVAPGKTARITCGRNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHVVFGGGTKLTVLGSRGGGGSGGGGSGGSLEMAEVQLVQSGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVRGRFTISRDNAKNSLYLQMNSLRAEDTALYYCARERGYGYHDPHDYWGQGTLVTVSS (SEQ ID NO: 90) DNAcagtctgtcgtgacgcagccgccctcggtgtcagtggccccaggaaagacggccaggattacctgtgggagaaacaacattggaagtaaaagtgtgcactggtaccagcagaagccaggccaggcccctgtgctggtcgtctatgatgatagcgaccggccctcagggatccctgagcgattctctggctccaactctgggaacacggccaccctgaccatcagcagggtcgaagccggggatgaggccgactattactgtcaggtgtgggatagtagtagtgatcatgtggtattcggcggagggaccaagctgaccgtcctaggttctagaggtggtggtggtagcggcggcggcggctctggtggatccctcgagatggccgaagtgcagctggtgcagtctgggggaggtgtggtacggcctggggggtccctgagactctcctgtgcagcctctgggttcacctttgatgattatggcatgagctgggtccgccaagctccagggaaggggctggagtgggtctctggtattaattggaatggtggtagcacaggttatgcagactctgtgaggggccgattcaccatctccagagacaacgccaagaactccctgtatctgcaaatgaacagtctgagagccgaggacacggccttgtattactgtgcgagagagcgtggctacgggtaccatgatccccatgactactggggccaaggcaccctggtgaccgtctcctca (SEQ ID NO: 91)

In another embodiment, the antibody or antigen binding protein is ananti-WT1 scFv or antigen binding fragment thereof that has an antigenbinding region that comprises the amino acid sequence of SEQ ID NO: 108and specifically binds to a peptide with the amino acid sequenceRMFPNAPYL (SEQ ID NO: 1) in conjunction with HLA-A0201. In otherembodiments, the anti-WT-1 antibody is a scFv-Fc fusion protein or fulllength human IgG with VH and VL regions or CDRs selected from Table 6.

TABLE 6 Antigen WT1 (Ext002 #23) Peptide RMFPNAPYL (SEQ ID NO. 1) CDRs:1 2 3 VH GFSVSGTYMG LLYSGGGTYHPASLQ GGAGGGHFDS (SEQ ID NO. 92) G(SEQ ID NO. 94) (SEQ ID NO. 93) DNA gggttctccgtcagtggcaccttctttatagtggtggcggcac gaggggcaggaggtggcc ctacatgggcataccacccagcgtccctgca actttgactcc (SEQ ID NO. 95) gggc (SEQ ID NO. 97)(SEQ ID NO. 96) VL TGSSSNIGAGYDVH GNSNRPS AAWDDSLNGYV (SEQ ID NO. 98)(SEQ ID NO. 99) (SEQ ID NO. 100) DNA actgggagcagctccaacggtaacagcaatcggccctca gcagcatgggatgacagcct atcggggcaggttatgatgt(SEQ ID NO. 102) gaatggttatgtc acac (SEQ ID NO. 103) (SEQ ID NO. 101)Full EVQLVETGGGLLQPGGSLRLSCAASGFSVSGTYMGWVRQAPGKGLE VHWVALLYSGGGTYHPASLQGRFIVSRDSSKNMVYLQMNSLKAEDTAVYYCAKGGAGGGHFDSWGQGTLVTVSS (SEQ ID NO. 104) DNAgaggtgcagctggtggagaccggaggaggcttgctccagccgggggggtccctcagactctcctgtgcagcctctgggttctccgtcagtggcacctacatgggctgggtccgccaggctccagggaagggactggagtgggtcgcacttctttatagtggtggcggcacataccacccagcgtccctgcagggccgattcatcgtctccagagacagctccaagaatatggtctatcttcaaatgaatagcctgaaagccgaggacacggccgtctattactgtgcgaaaggaggggcaggaggtggccactttgactcctggggccaaggcaccctggtgaccgtctcctca (SEQ ID NO. 105) FullQSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPK VLLLIYGNSNRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWD DSLNGYVFGTGTKLTVLG(SEQ ID NO. 106) DNAcagtctgtgttgacgcagccgccctcagtgtctggggccccagggcagagggtcaccatctcctgcactgggagcagctccaacatcggggcaggttatgatgtacactggtaccagcagcttccaggaacagcccccaaactcctcatctatggtaacagcaatcggccctcaggggtccctgaccgattctctggctccaagtctggcacctcagcctccctggccatcagtgggctccagtctgaggatgaggctgattattactgtgcagcatgggatgacagcctgaatggttatgtcttcggaactgggaccaagctgaccgtccta ggt(SEQ ID NO. 107) scFv QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWD DSLNGYVFGTGTKLTVLGSRGGGGSGGGGSGGGGSLEMAEVQLVETGGGLLQPGGSLRLSCAASGFSVSGTYMGWVRQAPGKGLEWVALLYSGGGTYHPASLQGRFIVSRDSSKNMVYLQMNSLKAEDTAVYYCAKGGAGGGHFDS WGQGTLVTVSS(SEQ ID NO. 108) DNAcagtctgtgttgacgcagccgccctcagtgtctggggccccagggcagagggtcaccatctcctgcactgggagcagctccaacatcggggcaggttatgatgtacactggtaccagcagcttccaggaacagcccccaaactcctcatctatggtaacagcaatcggccctcaggggtccctgaccgattctctggctccaagtctggcacctcagcctccctggccatcagtgggctccagtctgaggatgaggctgattattactgtgcagcatgggatgacagcctgaatggttatgtcttcggaactgggaccaagctgaccgtcctaggttctagaggtggtggtggtagcggcggcggcggctctggtggtggtggatccctcgagatggccgaggtgcagctggtggagaccggaggaggcttgctccagccgggggggtccctcagactctcctgtgcagcctctgggttctccgtcagtggcacctacatgggctgggtccgccaggctccagggaagggactggagtgggtcgcacttctttatagtggtggcggcacataccacccagcgtccctgcagggccgattcatcgtctccagagacagctccaagaatatggtctatcttcaaatgaatagcctgaaagccgaggacacggccgtctattactgtgcgaaaggaggggcaggaggtggccactttgactcctggggccaaggcaccctggtgaccgtctcctca (SEQ ID NO. 109)

Other embodiments of the disclosed antibodies and antigen bindingproteins encompass those comprising light and heavy hypervariableregions and constant regions, for example as shown in Tables 7 (heavychain), 8 (light chain) and 9 (constant regions).

TABLE 7 CDR- SEQ ID H1 CDR-H2 CDR-H3 NO. Group I EXT002-12(166) SNAVAWNRTYRGSTYY---ALSV G-SNSAFDF 119-121 EXT002-5(184) SNSAAWNRTYYGSKWYNDYAVSV GRLGDAFDI 122-124 EXT002-8(184) SDGAAWNRTYYRSKWYNDYAVSV GDYYYGMDV 125-127 Consensus(191) SNAAAWNRTYYGSKWYNDYAVSV G    AFDI 128-130 Group II EXT002-14(163) SYWISRIDPSDSYTNYSPSFQG GD------YDFYLDP-- 131-133 EXT002-25(163) SYGISWISAYNGNTNYAQKLQG DLYSSGWYESYYYGMDV 134-136 EXT002-3(186) SYAISGIIPIFGTANYAQKFQG RIP-P------YYGMDV 137-139 EXT002-30(163) SYGISWISAHNGNTNYAQKLQG DR-------VWFGDLSD 134, 140, 141 EXT002-33(163) SYAISGIIPIFGTANYAQKEQG NYDFWSG-----DAFDI 137, 142, 143 Consensus(188) SYAIS I P  G TNYAQKFQG           FY GMDV 137, 144, 145 Group IIIEXT002-34(161) DYGMS GINWNGGSTGYADSV ERGY-GYHDPHDY 146-148EXT002-40(157) NYTMN SISLSGAYIYYADSL EGYSSSVYDAFDL 149-151EXT002-45(165) SYGMH GILSDGGKDYYVDSV CSSN-YGNDAFDI 152-154EXT002-48(165) TYSMN SISSGAYSIFYADSV DQYYGDKWDAFDI 155-157Consensus(170) SYGMN SISS GGSIYYADSV E YY   WDAFDI 158-160

TABLE 8 SEQ ID CDR-L1 CDR-L2 CDR-L3 NOS. Group I EXT002-1 (46)CSGSSSNIGS-NTVN SNNQRPSG AAWDDSLNG--WVFG 161-163 EXT002-10 (46)CSGSSSNIGS-NTVN SNNQRPSG EAWDDSLKG--PVFG 161, 162, 164 EXT002-12 (22)CTGSSSNIGAGYDVH GNSNRPSG QSYDSSLSADNYVFG 165-167 EXT002-13 (46)CSGSSSNIGS-NTVN SNNQRPSG AAWDDSLNG--WVFG 161-163 EXT002-2 (46)CSGSSSNIGR-NIVN SNIERPSG ASWDDSLNG--VLFG 168-170 EXT002-20 (46)CSGSRSNIAS-NGVG KNDQRPSG SAWDDSLDGH-VVFG 171-173 EXT002-23 (46)CTGSSSNIGAGYDVH GNSNRPSG AAWDDSLNG--YVFG 165, 166, 174 EXT002-25 (22)CSGSSSNIGS-STVN SNSQRPSG AAWDDSLNG--VVFG 175-177 EXT002-3 (46)CSGSSSNIGS-NYVY RSNQRPSG AAWDDSLNG--VVFG 178, 179, 177 EXT002-30 (22)CSGSSSNIGR-NTVN SNNQRPSG AAWDDSLNG--YVFG 180, 162, 174 EXT002-33 (22)CSGSSSNIGN-DYVS DNNKRPSG GTWDNSLSA--WVFG 181-183 EXT002-36 (22)CSGSSSNIGS-NSVY NNNQRPSG ATWDDSLSG--WVFG 184-186 EXT002-40 (22)CSGSSSNIGS-NYVY RNNQRPSG AAWDDSLSA--WVFG 178, 187, 188 EXT002-42 (46)CSGSTSNIGS-YYVS DNNNRPSG GTWDSSLSA--WVFG 189-191 EXT002-45 (22)CSGSSSNIGN-NYVS DNNKRPSG GTWDSSLSA--WVFG 192, 182, 191 EXT002-48 (22)CSGSNSNIGT-NTVT SNFERPSG SAWDDSFNG--PVFG 193-195 EXT002-6 (46)CSGSSSNIGS-NYVS RNNQRPSG AAWDDGLRG--YVFG 196, 187, 197 EXT002-9 (22)CSGSSSNIGS-NTVN SNNQRPSG EAWDDSLKG--PVFG 161, 162, 164 Consensus (46)CSGSSSNIGS N V  NNQRPSG AAWDDSL G  WVFG 161-163 Group II EXT002-24 (24)RASQSISSYLN AASSLQS QQSYSTP--T 198-200 EXT002-31 (24) RASQGISNYLAAASTLQS QKYNSAPGVT 201-203 EXT002-35 (24) RASQSINGWLA RASTLQS QQSSSLP-FT204-206 EXT002-5 (48) RASQSISSYLN AASSLQS QQSYSTP-LT 198-200EXT002-7 (48) RASQGISYYLA AASTLKS QQLNSYP-LT 207-209 EXT002-B (48)RASQSISSYLN AASSLQS QQSYSTP-WT 198-200 Consensus (48) RASQSISSYLNAASSLQS QQSYSTP LT 198-200 Group III EXT002-16 (23) GGNNIGSKSVH DDSDRPSQVWDSSSDHPV 210-212 EXT002-17 (47) GGNNIGSKSVH DDSDRPS QVWDSSGDHPV 210,211, 213 EXT002-19 (47) GGNNIGSKSVH YDSDRPS QVWDSSSDHPV 210, 214, 212EXT002-21 (19) GGTNIGSRFVH DDSDRPS QVWDSSGDHPV 215, 211, 213EXT002-22 (47) GGNNVESKSVH YDRDRPS EVWDSGSDHPV 216-218 EXT002-32 (23)GGKNIGSKSVH YDSDRPS QVWDSGSDHYV 219, 214, 220 EXT002-34 (23) GGNNIGSKSVHDDSDRPS QVWISSGDRVI 210, 211, 221 EXT002-43 (23) GGDNIGSQGVH YDTDRPSQVWGASSDHPV 222-224 Consensus (47) GGNNIGSKSVH YDSDRPS QVWDSSSDHPV 210,214, 212 Group IV EXT002-11 (47) TGTSSDVGGYNYVS DVSKRPS GIYTYSDSW--V225-227 EXT002-14 (23) TGTSSDVGGYNYVS DVGNRPS SSYTSSSTR--V 225, 228, 229EXT002-26 (23) TGTRSDVGLYNYVA DVIYRPG SSYTNTGTV--L 230-232 EXT002-4 (47)TGTSSDFGDYDYVS DVSDRPS QSYDSSLSGSGV 233-235 Consensus (47)TGTSSDVGGYNYVS DVS RPS SSYTSS S   V 225, 234, 229

TABLE 9 Constant Regions Human   ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVheavy SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT chain  QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEL constantLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV regionKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD andWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL IgG1 FcPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN domainYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH sequence EALHNHYTQKSLSLSPGK (SEQ ID NO. 236) Human   TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ lightWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY chainEKHKVYACEVTHQGLSSPVTKSFNRGEC (kappa) (SEQ ID NO. 237) Human  QPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTV lightAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQW chainKSHRSYSCQVTHEGSTVEKTVAPTECS  (lambda) (SEQ ID NO. 238)

The invention relates to recombinant antigen-binding proteins,antibodies and antigen binding fragments thereof that specificallyrecognize epitopes of a complex of a peptide/protein fragment derivedfrom an intracellular protein, and an MHC class I molecule, for example,as the complex might be displayed at the cell surface during antigenpresentation. The heavy and light chains of an antibody of the inventionmay be full-length (e.g., an antibody can include at least one, andpreferably two, complete heavy chains, and at least one, and preferablytwo, complete light chains) or may include an antigen-binding portion (aFab, F(ab′)₂, Fv or a single chain Fv fragment (“scFv”)). In otherembodiments, the antibody heavy chain constant region is chosen from,e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE,particularly chosen from, e.g., IgG1, IgG2, IgG3, and IgG4, moreparticularly, IgG1 (e.g., human IgG1). In another embodiment, theantibody light chain constant region is chosen from, e.g., kappa orlambda, particularly kappa.

The antibodies and antigen binding proteins of the present invention areintended to encompass bispecific antibodies, including bispecific T-cellengaging antibodies, that is, antibodies comprising two antibodyvariable domains on a single polypeptide chain that are able to bind twoseparate antigens. Where a first portion of a bispecific antibody bindsan antigen on a tumor cell for example and a second portion of abispecific antibody recognizes an antigen on the surface of a humanimmune effector cell, the antibody is capable of recruiting the activityof that effector cell by specifically binding to the effector antigen onthe human immune effector cell. In some instances, bispecificantibodies, therefore, are able to form a link between effector cells,for example, T cells and tumor cells, thereby enhancing effectorfunction.

In one embodiment, the constant region/framework region is altered, forexample, by amino acid substitution, to modify the properties of theantibody (e.g., to increase or decrease one or more of: antigen bindingaffinity, Fc receptor binding, antibody carbohydrate, for example,glycosylation, fucosylation etc, the number of cysteine residues,effector cell function, effector cell function, complement function orintroduction of a conjugation site). Furthermore, conjugation of theantibody to a drug, toxin, radioisotope, cytokine, inflammatory peptideor cytotoxic agent is also contemplated.

In one embodiment, the antibody is an anti-WT1/A2 antibody and comprisesthe human IgG1 constant region and Fc domain shown in Table 9. In oneembodiment, the anti-WT1/A2 antibody comprises a human kappa sequence,or a human lambda sequence having the sequence set forth in Table 9. Theamino acid sequences for some complementarity determining regions (CDRs)of antibodies of the invention are shown in Tables 1-8.

The present invention is based on the identification of antigen-specificbinding sequences from which a variety of antigen-binding proteins canbe produced. In addition to antibodies specific for an antigen thatrepresents a protein fragment (peptide)/HLA complex similar to thattypically recognized by a T-cell receptor following antigen processingand presentation of the protein to the T-cell, identification of aminoacid and nucleic sequences as disclosed herein for the preparation ofantibodies can also be used to generate other antigen-binding moleculesincluding chimeric antigen receptors (CARs), with specificity for theprotein fragment (peptide)/HLA complex. These can be incorporated intocells to make them specifically cytotoxic to the antigen expressingcell.

The present invention employs a novel approach to obtaining therapeuticantibodies to any protein, including those proteins that areinaccessible because they are not expressed on the cell surface. Nearlyany intracytoplasmic or intranuclear protein (in addition to cellsurface proteins) is a potential target for the approach describedherein. This includes, but is not limited to, oncogenic proteins,transcription factors, enzymes, etc.

In order to target tumor antigens derived from intracellular or nuclearproteins, development of a therapeutic antibody an uncommon approach wasrequired. This approach is to generate recombinant mAbs that recognizethe peptide/MHC complex expressed on the cell surface, with the samespecificity as a T-cell receptor (TCR). Such mAbs share functionalhomology with TCRs regarding target recognition, but confer higheraffinity and capabilities of arming with potent cytotoxic agents thatantibodies feature. Technically, TCR-like mAbs may be generated byconventional hybridoma techniques known to those of skill in the art, toproduce human, humanized or chimeric antibodies.

Furthermore, fully-human mAbs are preferred for therapeutic use inhumans because murine antibodies cause an immunogenicity reaction, knownas the HAMA (human anti-mouse antibodies) response (24, 25), whenadministered to humans, causing serious side effects, includinganaphylaxis and hypersensitivity reactions. This immunogenicity reactionis triggered by the human immune system recognizing the murineantibodies as foreign because of slightly different amino acid sequencesfrom natural human antibodies. Humanization methods known in the art(26, 27) can be employed to reduce the immunogenicity of murine-derivedantibodies (28).

Recently, the use of phage display libraries has made it possible toselect large numbers of Ab repertoires for unique and rare Abs againstvery defined epitopes (for more details on phage display see McCaffertyet al., Phage antibodies: filamentous phage displaying antibody variabledomains. Nature, 348: 552-554.) The rapid identification of human Fab orsingle chain Fv (ScFV) fragments highly specific for tumorantigen-derived peptide-MHC complex molecules has thus become possible(19-22). More recently, immuno-toxins, generated by fusing TCR-like Fabspecific for melanoma Ag MART-1 26-35/A2 or gp100 280-288/A2 to atruncated form of Pseudomonas endotoxin, have been shown to inhibithuman melanoma growth both in vitro and in vivo (23). In addition, byengineering full-length mAb using the Fab fragments, it is possible todirectly generate a therapeutic human mAb, by-passing months oftime-consuming work, normally needed for developing therapeutic mAbs.The present invention involves the development of a TCR-like, fullyhuman mAb that recognizes, for example, the WT1 peptide/HLA-A2 complex(RMFPNAPYL, SEQ ID NO: 1) for cancer therapy.

Recombinant antibodies with TCR-like specificity represent a new andvaluable tool for research and therapeutic applications in tumorimmunology and immunotherapy. WT1 is a well-established and validatedtumor antigen that has been investigated throughout the world as amarker, prognostic factor and therapeutic target. It was recentlyprioritized as the top priority tumor antigen by an NCI task force (29).

Identification of Peptides with High Predictive Binding to HLA Molecules

In one embodiment, the present invention relates to a method for thegeneration of antibodies that specifically bind to HLA-restrictedpeptides, which, when presented as part of a peptide/MHC complex areable to elicit a specific cytotoxic T-cell response. HLA class Imolecules present endogenous derived peptides of about 8-12 amino acidsin length to CD8⁺ cytotoxic T lymphocytes. Peptides to be used in themethod of the present invention are generally about 6-22 amino acids inlength, and in some embodiments, between about 9 and 20 amino acids andcomprise an amino acid sequence derived from a protein of interest, forexample, human WT1 protein (Genbank accession no. P19544) or an analogthereof.

Peptides suitable for use in generating antibodies in accordance withthe method of the present invention can be determined based on thepresence of HLA-A0201-binding motifs and the cleavage sites forproteasomes and immune-proteasomes using computer prediction modelsknown to those of skill in the art. For predicting MHC class I bindingsites, such models include, but are not limited to, ProPred1 (describedin more detail in Singh and Raghava, ProPred: prediction of HLA-DRbinding sites. BIOINFORMATICS 17(12):1236-1237 2001), and SYFPEITHI (seeSchuler et al. SYFPEITHI, Database for Searching and T-Cell EpitopePrediction. in Immunoinformatics Methods in Molecular Biology, vol409(1): 75-93 2007)

HLA-A*0201 is expressed in 39-46% of all caucasians and therefore,represents a suitable choice of MHC antigen for use in the presentmethod. For preparation of one embodiment of a WT1 peptide antigen,amino acid sequences and predicted binding of putative CD84⁺ epitopes toHLA-A0201 molecules were identified using the predictive algorithm ofthe SYFPEITHI database (see Schuler et al. SYFPEITHI, Database forSearching and T-Cell Epitope Prediction. in Immunoinformatics Methods inMolecular Biology, vol 409(1): 75-93 2007).

Once appropriate peptides have been identified, peptide synthesis may bedone in accordance with protocols well known to those of skill in theart. Because of their relatively small size, the peptides of theinvention may be directly synthesized in solution or on a solid supportin accordance with conventional peptide synthesis techniques. Variousautomatic synthesizers are commercially available and can be used inaccordance with known protocols. The synthesis of peptides in solutionphase has become a well-established procedure for large-scale productionof synthetic peptides and as such is a suitable alternative method forpreparing the peptides of the invention. (See for example, Solid PhasePeptide Synthesis by John Morrow Stewart and Martin et al. Applicationof Almez-mediated Amidation Reactions to Solution Phase PeptideSynthesis, Tetrahedron Letters Vol. 39, pages 1517-1520 1998.)

Each of the peptides used in the protocols described herein waspurchased and synthesized by Genemed Synthesis, Inc. (San Antonio, Tex.)using fluorenylmethoxycarbonyl chemistry and solid-phase synthesis andpurified by high-pressure liquid chromatography. The quality of thepeptides was assessed by high-performance liquid chromatographyanalysis, and the expected molecular weight was observed usingmatrix-assisted laser desorption mass spectrometry. Peptides weresterile and 70% to 90% pure. The peptides were dissolved in DMSO anddiluted in PBS (pH 7.4) or saline at 5 mg/mL and stored at −80° C.

Subsequent to peptide selection, binding activity of selected peptidesis tested using the antigen-processing-deficient T2 cell line, whichincreases expression of HLA-A when stabilized by a peptide in theantigen-presenting groove. Briefly, T2 cells are pulsed with peptide fora time sufficient to induce HLA-A expression. HLA-A expression of T2cells is then measured by immunostaining with a fluorescently labeledmonoclonal antibody specific for HLA-A (for example, BB7.2) and flowcytometry. Fluorescence index (FI) is calculated as the meanfluorescence intensity (MFI) of HLA-A0201 on T2 cells as determined byfluorescence-activated cell-sorting analysis, using the formulaFI=(MFI[T2 cells with peptide]/MFI [T2 cells without peptide]−1.

Fully human T-cell receptor (TCR)-like antibodies to Wilm's tumoroncognene protein (WT1) were produced using the method disclosed herein.TCR-like anti-WT1 antibodies generated by phage display technology arespecific for a WT1 peptide/HLA complex similar to that which inducesHLA-restricted cytotoxic CD8 T-cells.

The WT1 protein sequence was screened using the SYFPEITHI algorithm andWT1 peptides (for example, peptides designated 428, 328, and 122) wereidentified that had predicted high-affinity binding to multiple HLAmolecules that are highly expressed in the Caucasian population. Peptide428 spans WT1 amino acids 428-459, peptide 328 spans WT1 amino acids328-349, and peptide 122 spans WT1 amino acids 122-140 (see FIG. 1)

Heteroclitic peptides can also be designed by conservative amino acidsubstitutions of MHC-binding residues expected to enhance the affinitytoward the MHC class 1 allele, as predicted by the prediction algorithm.WT1 peptide 122 comprises within it a known CD8⁺ epitope (126-134). Inone embodiment, therefore, a modified peptide of the peptide that spansthe WT1 amino acid residues 126-134 and contains a modified amino acidat positions may be used. Peptides used for alanine mutagenesis of WT1A(otherwise designated RFM) were named based on the position where thesubstitution was made. Examples of WT1 peptides which may be used areshown in Table 10 along with irrelevant peptides, RHAMM-R3 and EW.

TABLE 10 WT1A (RMF) RMFPNAPYL SEQ ID NO.: 1 WT1A1-B AMFPNAPYLSEQ ID NO.: 110 WT1A-3 RMAPNAPYL SEQ ID NO.: 111 WT1A-4 RMFANAPYLSEQ ID NO.: 112 WT1A-5 RMFPAAPYL SEQ ID NO.: 113 WT1A-7 RMFPNAAYLSEQ ID NO.: 114 WT1A-8 RMFPNAPAL SEQ ID NO.: 115 RHAMM-R3 ILSLELMKLSEQ ID NO.: 116 EW QLQNPSYDK SEQ ID NO.: 117 RSDELVRHHNMHQRNMTKLSEQ ID NO.: 118 PGCNKRYFKLSHLQMHSRKHTG SEQ ID NO.: 119SGQARMFPNAPYLPSCLES SEQ ID NO.: 120 SGQAYMFPNAPYLPSCLES SEQ ID NO.: 121

Once a suitable peptide has been identified, the target antigen to beused for phage display library screening, that is, a peptide/HLA complex(for example, WT1 peptide/HLA-A0201) is prepared by bringing the peptideand the histocompatibility antigen together in solution to form thecomplex.

Selecting a High Affinity ScFV Against a WT1 Peptide

The next step is the selection of phage that bind to the target antigenof interest with high affinity, from phage in a human phage displaylibrary that either do not bind or that bind with lower affinity. Thisis accomplished by iterative binding of phage to the antigen, which isbound to a solid support, for example, beads or mammalian cells followedby removal of non-bound phage and by elution of specifically boundphage. In one embodiment, antigens are first biotinylated forimmobilization to, for example, streptavidin-conjugated Dynabeads M-280.The phage library is incubated with the cells, beads or other solidsupport and non binding phage is removed by washing. Clones that bindare selected and tested.

Once selected, positive scFv clones are tested for their binding toHLA-A2/peptide complexes on live T2 cell surfaces by indirect flowcytometry. Briefly, phage clones are incubated with T2 cells that havebeen pulsed with Wt1-A peptide, or an irrelevant peptide (control). Thecells are washed and then with a mouse anti-M13 coat protein mAb. Cellsare washed again and labeled with a FITC-goat (Fab)₂ anti-mouse Ig priorto flow cytometry.

In other embodiments, the anti-WT1/A antibodies may comprise one or moreframework region amino acid substitutions designed to improve proteinstability, antibody binding, expression levels or to introduce a sitefor conjugation of therapeutic agents. These scFv are then used toproduce recombinant human monoclonal Igs in accordance with methodsknown to those of skill in the art.

Methods for reducing the proliferation of leukemia cells is alsoincluded, comprising contacting leukemia cells with a WT1 antibody ofthe invention. In a related aspect, the antibodies of the invention canbe used for the prevention or treatment of leukemia. Administration oftherapeutic antibodies is known in the art.

Antibody Conjugates with Anti-Cancer Agents

Monoclonal antibodies represent the preferred vehicle for the targeteddelivery of bioactive agents to cancer sites, including antibody-baseddelivery of cytotoxics, radionuclides or immunomodulatory cytokines.Conjugates of the antibodies of the invention with therapeutic agents,including without limitation, drugs (such as calecheamicin, aureastatin,doxorubicin), or toxins (such as ricin, diphtheria, gelonin) orradioisotopes emitting alpha or beta particles (such as, ⁹⁰Y, ¹³¹I,²²⁵Ac, ²¹³Bi, ²²³Ra and ²²⁷Th), inflammatory peptides (such as IL2, TNF,IFN-γ) are encompassed by the invention.

Pharmaceutical Compositions and Methods of Treatment

WT1 antibodies of the present invention can be administered fortherapeutic treatments to a patient suffering from a tumor orWT1-associated pathologic condition in an amount sufficient to prevent,inhibit, or reduce the progression of the tumor or pathologic condition.Progression includes, e.g, the growth, invasiveness, metastases and/orrecurrence of the tumor or pathologic condition. Amounts effective forthis use will depend upon the severity of the disease and the generalstate of the patient's own immune system. Dosing schedules will alsovary with the disease state and status of the patient, and willtypically range from a single bolus dosage or continuous infusion tomultiple administrations per day (e.g., every 4-6 hours), or asindicated by the treating physician and the patient's condition.

The identification of medical conditions treatable by WT1 antibodies ofthe present invention is well within the ability and knowledge of oneskilled in the art. For example, human individuals who are eithersuffering from a clinically significant leukemic disease or who are atrisk of developing clinically significant symptoms are suitable foradministration of the present WT1 antibodies. A clinician skilled in theart can readily determine, for example, by the use of clinical tests,physical examination and medical/family history, if an individual is acandidate for such treatment.

Non-limiting examples of pathological conditions characterized by WT1expression include chronic myelocytic leukemia, acute lymphoblasticleukemia (ALL), acute myeloid/myelogenous leukemia (AML) andmyelodysplastic syndrome (MDS). Additionally, solid tumors, in generaland in particular, tumors associated with mesothelioma, ovarian cancer,gastrointestinal cancers, breast cancer, prostate cancer andglioblastoma are amenable to treatment using WT1 antibodies.

In another embodiment, therefore, the present invention provides amethod of treating a medical condition by administering a WT1 antibodyof the present invention in combination with one or more other agents.For example, an embodiment of the present invention provides a method oftreating a medical condition by administering a WT1 antibody of thepresent invention with an antineoplastic or antiangiogenic agent. TheWT1 antibody can be chemically or biosynthetically linked to one or moreof the antineoplastic or antiangiogenic agents.

Any suitable method or route can be used to administer a WT1 antibody ofthe present invention, and optionally, to coadminister antineoplasticagents and/or antagonists of other receptors. Routes of administrationinclude, for example, oral, intravenous, intraperitoneal, subcutaneous,or intramuscular administration. It should be emphasized, however, thatthe present invention is not limited to any particular method or routeof administration.

It is noted that a WT1 antibody of the present invention can beadministered as a conjugate, which binds specifically to the receptorand delivers a toxic, lethal payload following ligand-toxininternalization.

It is understood that WT1 antibodies of the invention will beadministered in the form of a composition additionally comprising apharmaceutically acceptable carrier. Suitable pharmaceuticallyacceptable carriers include, for example, one or more of water, saline,phosphate buffered saline, dextrose, glycerol, ethanol and the like, aswell as combinations thereof. Pharmaceutically acceptable carriers mayfurther comprise minor amounts of auxiliary substances such as wettingor emulsifying agents, preservatives or buffers, which enhance the shelflife or effectiveness of the binding proteins. The compositions of theinjection may, as is well known in the art, be formulated so as toprovide quick, sustained or delayed release of the active ingredientafter administration to the mammal.

Other aspects of the invention include without limitation, the use ofantibodies and nucleic acids that encode them for treatment of WT1associated disease, for diagnostic and prognostic applications as wellas use as research tools for the detection of WT1 in cells and tissues.Pharmaceutical compositions comprising the disclosed antibodies andnucleic acids are encompassed by the invention. Vectors comprising thenucleic acids of the invention for antibody-based treatment by vectoredimmunotherapy are also contemplated by the present invention. Vectorsinclude expression vectors which enable the expression and secretion ofantibodies, as well as vectors which are directed to cell surfaceexpression of the antigen binding proteins, such as chimeric antigenreceptors.

Cells comprising the nucleic acids, for example cells that have beentransfected with the vectors of the invention are also encompassed bythe disclosure.

For use in diagnostic and research applications, kits are also providedthat contain a WT1 antibody or nucleic acids of the invention, assayreagents, buffers, and the like.

The method of the present invention will now be described in more detailwith respect to representative embodiments.

Materials

Cell Samples, Cell Lines and Antibodies.

After informed consent on Memorial Sloan-Kettering Cancer CenterInstitutional Review Board approved protocols, peripheral bloodmononuclear cells (PBMC) from HLA-typed healthy donors and patients wereobtained by Ficoll density centrifugation. The sources for obtaininghuman mesothelioma cell lines are described previously (29). The celllines include: H-Meso1A, JMN, VAMT, H2452, H2373, H28, MSTO, Meso 11,Meso 34, Meso 37, Meso 47, and Meso 56. All cells were HLA typed by theDepartment of Cellular Immunology at Memorial Sloan-Kettering CancerCenter. Leukemia cell lines LAMA81, BV173, and 697, (WT1+, A0201+) werekindly provided by Dr. H. J. Stauss (University College London, London,United Kingdom). Melanoma cell line MeWo (WT1−, A201+), SKLY16 (B-celllymphoma; WT1−, A0201+); K562, RwLeu4, and HL60, all WT1+ leukemias, anda TAP-deficient T2 cell line were obtained from the American TypeCulture Collection. The cell lines were cultured in RPMI 1640supplemented with 5% FCS, penicillin, streptomycin, 2 mmol/L glutamine,and 2-mercaptoethanol at 37 C/5% CO2.

Monoclonal Ab against human HLA-A2 (clone BB7.2) conjugated to FITC orAPC, and its isotype control mouse IgG2b/FITC or APC, to human or mouseCD3, CD19, CD56, CD33, CD34 (BD Biosciences, San Diego), goat F(ab)2anti-human IgG conjugated with PE or FITC and goat F(ab)2 anti-mouseIg's conjugated to fluorescent (In Vitrogen, City) were purchased. MousemAb to HLA-class I (W6/32) was obtained from the MSKCC Monoclonalantibody Core Facility.

Peptides.

All peptides were purchased and synthesized by Genemed Synthesis, Inc.(San Antonio, Tex.). Peptides were >90% pure. (Table 1.) The peptideswere dissolved in DMSO and diluted in saline at 5 mg/mL and frozen at−180 C. Biotinylated single chain WT1 peptide/HLA-A0201 andRHAMM-3/HLA-A0201 complexes were synthesized by refolding the peptideswith recombinant HLA-A2 and beta2 microglobulen (β2M) at the Tetramerfacility at MSKCC.

Animals.

Eight to 10 week-old NOD.Cg-Prkdc scid IL2rgtm1WjI/SzJ mice, known asNOD scid gamma (NSG), were purchased from the Jackson Laboratory (BarHarbor, Me.) or obtained from MSKCC animal breeding facility.

Methods

Flow Cytometry Analysis.

For cell surface staining, cells were incubated with appropriate mAbsfor 30 minutes on ice, washed, and incubated with secondary antibodyreagents when necessary. Flow cytometry data were collected on a FACSCalibur (Becton Dickinson) and analyzed with FlowJo V8.7.1 and 9.4.8software.

Selection and Characterization of scFv Specific for WT1Peptide/HLA-A0201 Complexes.

A human scFv antibody phage display library was used for the selectionof mAb clones. In order to reduce the conformational change of MHC1complex introduces by immobilizing onto plastic surfaces, a solutionpanning method was used in place of conventional plate panning. Inbrief, biotinylated antigens were first mixed with the human scFv phagelibrary, then the antigen-scFv antibody complexes were pulled down bystreptavidin-conjugated Dynabeads M-280 through a magnetic rack. Boundclones were then eluted and were used to infect E. Coli XL1-Blue. ThescFv phage clones expressed in the bacteria were purified (35, 36).Panning was performed for 3-4 cycles to enrich scFv phage clones bindingto HLA-A0201/WT1 complex specifically. Positive clones were determinedby standard ELISA method against biotinylated single chain HLA-A0201/WT1peptide complexes. Positive clones were further tested for their bindingto HLA-A2/peptide complexes on live cell surfaces by flow cytometry,using a TAP-deficient, HLA-A0201+ cell line, T2. T2 cells were pulsedwith peptides (50 ug/ml) in the serum-free RPM11640 medium, in thepresence of 20 μg/ml β2 M ON. The cells were washed, and the stainingwas performed in following steps.

The cells were first stained with purified scFv phage clones, andfollowed by staining with a mouse anti-M13 mAb, and finally the goatF(ab)2 anti-mouse Ig's conjugate to FITC. Each step of the staining wasdone between 30-60 minutes on ice and the cells were washed twicebetween each step of the staining.

Engineering Full Length mAb Using the Selected ScFv Fragments.

Full-length human IgG1 of the selected phage clones were produced inHEK293 and Chinese hamster ovary (CHO) cell lines, as described (37). Inbrief, antibody variable regions were subcloned into mammalianexpression vectors, with matching human lambda or kappa light chainconstant region and human IgG1 constant region sequences. Molecularweight of the purified full length IgG antibodies were measured underboth reducing and non-reducing conditions by electrophoresis.

Engineering Chimeric Antigen Receptors and Immune Effector Cells.

Nucleic acids that encode antibodies and antigen binding proteinsidentified herein can be used engineer recombinant immune effectorcells. Methods and vectors to generate genetically modified T-cells, forexample, are known in the art (See Brentjens et al., Safety andpersistence of adoptively transferred autologous CD19-targeted T cellsin patients with relapsed or chemotherapy refractory B-cell leukemias inBlood 118(18):4817-4828, November 2011).

Characterization of the Full-Length Human IgG1 for the WT1p/A2 Complex.

Initially, specificities of the fully human IgG1 mAbs for the WT1peptide/A2 complex were determined by staining T2 cells pulsed with orwithout RMF or RHAMM-R3 control peptides, followed by secondary goatF(ab)2 anti-human IgG mAb conjugate to PE or FITC. The fluorescenceintensity was measured by flow cytometry. The same method was used todetermine the binding of the mAbs to fresh tumor cells and cell lines.

Radioimmunoassays.

WT1 ab1 was labeled with 125-I (Perkin Elmer) using the chloramine-Tmethod (38). 100 μg antibody was reacted with 1 mCi 125-I and 20 μgchloramine-T, quenched with 200 μg Na metabisulfite, then separated fromfree 125-I using a 10DG column (company) equilibrated with 2% bovineserum albumin in PBS. Specific activities of products were in the rangeof 7-8 mCi/mg.

Hematopoietic cell lines, adherent cell lines (harvested with anon-enzymatic cell stripper (name)), PBMCs from normal donors and AMLpatients were obtained as described. Cells were washed once with PBS andre-suspended in 2% human serum in PBS at 10⁷ cells/mL at 0°. Cells (10⁶tube in duplicate) were incubated with 125-I-labeled WT1 AB1 (1 μg/mL)for 45 minutes on ice, then washed extensively with 1% bovine serumalbumin in PBS at 0°. To determine specific binding, a duplicate set ofcells was assayed after pre-incubation in the presence of 50-fold excessunlabeled WT1 AB1 for 20 minutes on ice. Bound radioactivity wasmeasured by a gamma counter, specific binding was determined, and thenumber of bound antibodies per cell was calculated from specificactivity.

Antibody-Dependent Cellular Cytotoxicity (ADCC).

Target cells used for ADCC were T2 cells pulsed with or without WT1 orRHAAM-3 peptides, and tumor cell lines without peptide pulsing. WT1 ab1or its isotype control human IgG1 at various concentrations wereincubated with target cells and fresh PBMCs at different effector:target (E:T) ratio for 16 hrs. The supernatant were harvested and thecytotoxicity was measured by LDH release assay using Cytotox 96non-radioreactive kit from Promega following their instruction.Cytotoxicity is also measured by standard 4 hours 51Cr-release assay.

Transduction and selection of luciferase/GFP positive cells. BV173 andJMN cells were engineered to express high level of GFP-luciferase fusionprotein, using lentiviral vectors containing a plasmid encoding theluc/GFP (39). Using single cell cloning, only the cells showing highlevel GFP expression were selected by flow cytometry analysis and weremaintained and used for the animal study.

Therapeutic Trials of the WT1 Ab 1 in a Human Leukemia Xenograft NSGModel.

Two million BV173 human leukemia cells were injected IV into NSG mice.On day 5, tumor engraftment was confirmed by firefly luciferase imagingin all mice that were to be treated; mice were then randomly dividedinto different treatment groups. On day 6 and day 10, mAb WT1 ab1 or theisotype control mAb were injected IV. In animals that also receivedhuman effector cells with or without mAb, cells (CD34 and CD3-depletedhealthy donor human PBMCs) were injected IV into mice (10⁷ cells/mouse)4 hr before the mAb injections. Tumor growth was assessed byluminescence imaging once to twice a week, and clinical activity wasassessed daily.

Selection and Characterization of scFv Specific for WT1Peptide/HLA-A0201 Complexes.

Selection of an WT1-specific scFV is achieved using a 9-mer WT1-derivedpeptide comprising amino acids 126-134 (RMFPNAPYL, SEQ ID NO: 1) of WT1.This peptide has been shown to be processed and presented by HLA-A0201to induce cytotoxic CD8⁺ T cells that are capable of killingWT1-positive tumor cells.

Representative data from a patient with AML after 6 vaccinations with aWT1 RMF peptide are shown in FIG. 2 as evidence that the WT1 peptidesare immunogenic in humans. CD3 T cells were stimulated with WT1-Apeptide (amino acids 126-134) and cytotoxicity was measured using astandard ⁵¹Cr release assay against 697 (A0201⁺WT1⁺) or SKLY-16(A0201⁺WT1⁻) cell lines. The SKLY-16 cells pulsed with WT1-A orirrelevant peptide EW were used as positive and negative controls forthe specificity of the killing. Effector: Target (E:T) ratios areindicated on the X-axis. Data demonstrates that T cells killed WT1⁺tumor cells in a HLA-A0201-restricted manner.

Well established phage display libraries and screening methods known tothose of skill in the art were used to select scFv fragments highlyspecific for an WT1-A peptide/HLA-A2 complex. In one embodiment, a humanscFv antibody phage display library (7×10¹⁰ clones) was used for theselection of mAb clones. In order to reduce the conformational change ofMHC1 complex introduced by immobilizing onto plastic surfaces, asolution panning method was used in place of conventional plate panning.In brief, biotinylated antigens were first mixed with the human scFvphage library, then the antigen-scFv phage antibody complexes werepulled down by streptavidin-conjugated Dynabeads M-280 through amagnetic rack.

Bound clones were then eluted and were used to infect E. Coli XL1-Blue.The scFv phage clones expressed in the bacteria were purified (35, 36).Panning was performed for 3-4 cycles to enrich scFv phage clones bindingto HLA-A0201/WT1 complex specifically. Positive clones were determinedby standard ELISA method against biotinylated single chain HLA-A0201/WT1peptide complexes (FIG. 3). Positive clones were further tested fortheir binding to HLA-A2/peptide complexes on live cell surfaces by flowcytometry, using a TAP-deficient, HLA-A0201⁺ cell line, T2. T2 cellswere pulsed with peptides (50 μm/ml) in serum-free RPM11640 medium, inthe presence of 20 μg/ml 2 M overnight. The cells were washed, andstaining was performed as follows.

The cells were first stained with purified scFv phage clones, followedby staining with a mouse anti-M13 mAb, and finally, a goat F(ab)2anti-mouse lg conjugated to FITC. Each step of the staining was done for30-60 minutes on ice. The cells were washed twice between each stainingstep. Results are shown in FIG. 4. The phage clone of WT1 ab1 was shownto bind to T2 cells pulsed with only WT1-A peptide (RMFPNAPYL:abbreviated hereinafter as RMF), but not to T2 cells alone, T2 cellspulsed with control EW peptide, or heteroclitic peptide WT1-A.

Binding affinity of the full-length lgG1 of WT1 ab1 to the peptide/A0201complex was tested by titration of WT1 ab1 at indicated concentrations.T2 cells were pulsed with 50 μg/ml or 10 μg/ml, followed by secondarygoat F(ab) anti-human lgG/PE. Results are shown in FIG. 5.

FIG. 6 shows the density of peptide/HLA-A0201 complex recognized by WT1ab. T2 cells were pulsed overnight (ON) with RMF (upper panel) orRHAMM-R3 (lower panel) peptides at indicated concentrations, and bindingof WT1 ab1, WT1 ab3 and WT1 ab5 at a concentration of 1 μg/ml wasanalyzed by flow cytometry.

TABLE 11 Summary of phage panning for WTI/A2 Number Solution of singleELISA Number Rounds of clones positive of Unique Phage Libraries Panningscreened Rate Clones scFv-spleen A 4 72 41/96  13 scFv-spleen B 4 473/47 2 scFv-spleen C 3 58 0/58 0 scFv-PBMCA 4 68 34/68  10 scFv-PBMC B 390 19/90  7 Fab-spleen A 4 12 2/12 0 Fab-spleen B 4 36 0/36 0 Fab-spleenC 4 24 2/24 1 Fab-spleen C 3 72 38/72  5 Fab-spleen D 4 72 4/72 1Fab-spleen D 4 72 4/72 3

The positive scFv clones were tested for their binding to HLA-A2/peptidecomplexes on live cell surfaces by indirect flow cytometry on: (i) a TAPdeficient HLA-A0201+ T2 cells pulsed with WT1 peptide or irrelevantpeptide; (ii) a WT1+ HLAA0201+ cell lines such as BV173, U266 andcontrol WTr HLA-A0201+ cell line SKLY16, or WT1+ HLA-A0201-cell line,K562, without pulsing with the peptide. The latter determine therecognition and binding affinity of the scFv to the naturally processedWT1p/A2 complex on tumor cells.

A total of 28 phage clones were screened for their ability to producemAb specific for the WTI-A peptide/A2 complex. The recognition of theWT1p/A2 complex on live cells was measured by the binding of the phagescFv to T2 cells pulsed with the WTI-A peptide and the otherHLA-A2-binding peptides (50 μg/ml). These include: T2 cells alone; T2cells pulsed with WTI-A peptide; T2 cells pulsed with heterocliticpeptide WT1-A1; T2 cells pulsed with irrelevant EW peptide(HLA-A0201-binding 9-mer peptide, derived from Ewing sarcoma) orRHAMM-R3 (FIG. 4).

TABLE 12 Positive for binding toT2 Selected for Construction Clone #pulsed with WT1A of full-length IgG1 1 + 2 + 3 + + 4 + 5 + + 6 + 7 8 + 910 11 12 13 + + 14 15 + + 16 17 + 18 + + 19 + 20 + 21 22 + 23 + + 24-28Engineering Full Length mAb Using the Selected ScFv Fragments.

Phage display technology allows for the rapid selection and productionof antigen-specific scFv and Fab fragments, which are useful in and ofthemselves, or which can be further developed to provide completeantibodies, antigen binding proteins or antigen binding fragmentsthereof. Complete mAbs with Fc domains have a number of advantages overthe scFv and Fab antibodies. First, only full length Abs exertimmunological function such as CDC and ADCC mediated via Fc domain.Second, bivalent mAbs offer stronger antigen-binding affinity thanmonomeric Fab Abs. Third, plasma half-life and renal clearance will bedifferent with the Fab and bivalent mAb. The particular features andadvantages of each can be matched to the planned effector strategy.Fourth, bivalent mAb may be internalized at different rates than scFvand Fab, altering immune function or carrier function. Alpha emitters,for example, do not need to be internalized to kill the targets, butmany drugs and toxins will benefit from internalization of the immunecomplex. In one embodiment, therefore, once scFv clones specific forWT1p/A2 were obtained from phage display libraries, a full length IgGmAb using the scFv fragments was produced.

To produce recombinant human monoclonal IgG in Chinese hamster ovary(CHO) cells, a full length IgG mAb was engineered based on a methodknown to those of skill in the art (Tomomatsu et al., Production ofhuman monoclonal antibodies against FceRIa by a method combining invitro immunization with phage display. Biosci Biotechnol Biochem 73(7):1465-1469 2009). Briefly, antibody variable regions were subcloned intomammalian expression vectors (FIG. 7), with matching Lambda or Kappalight chain constant sequences and IgG1 subclass Fc (for example, seeTable 9) (33,34). Purified full length IgG antibodies showed expectedmolecular weight under both reducing and non-reducing conditions (FIG.8). Kinetic binding analysis (35) confirmed specific binding of fulllength IgG to WT1/A2, with a KD in nanomolar range (FIGS. 9 and 10.)

Example 1

Selection of ScFv Specific for WT1p/A2 Complex Using a Fully Human PhageDisplay Library.

Phage display against HLA-A0201/WT1 peptide complex was performed for3-4 panning rounds to enrich the scFv phage clones binding toHLA-A0201/WT1 peptide complex specifically. Individual scFv phage clonespositive for the WT1 peptide/A2 complex were determined by ELISA and theclones that possessed unique DNA coding sequences were subjected tofurther characterization. To test if the ScFv bound to the WT1p/A2complex on live cells, the positive phage clones were tested for bindingto a TAP deficient, HLA-A0201-positive cell line, T2. T2 cells can onlypresent the exogenous peptides and therefore have been widely used fordetection of specific epitopes presented by HLA-A2 molecules. A total 35phage clones were screened on T2 cells and 15 clones showed specificbinding to T2 cells pulsed with only WT1 RMF peptide, but not to T2cells alone or pulsed with control RHAMM-3 peptide (FIG. 4). The scFvphage clones were unable to bind to several tumor cell lines that areWT1− and HLA-A2 positive suggesting the affinity of the ScFv was weak,compared to full-length bivalent mAb.

Example 2

Generation of Full-Length Human IgG1.

Immunological function such as CDC and ADCC depend on the Fc domain ofbivalent IgG. In addition, bivalent mAbs offer stronger antigen-bindingavidity than monomeric scFv Abs. Therefore, 6 ScFv phage clones among 15positive phage clones were selected to produce the full-length humanmonoclonal IgG1 in HEK293 and Chinese hamster ovary (CHO) cells. Inbrief, variable regions of the mAbs were subcloned into mammalianexpression vectors with matching human lambda or kappa light chainconstant region and human IgG1 constant region sequences. Purified fulllength IgG antibodies showed expected molecular weight under bothreducing and non-reducing conditions (FIG. 8). Five clones weresuccessfully engineered into human IgG1.

Example 3

Specificity and Binding Avidity of the IgG1 mAb

Binding to Human Cell Lines.

T2 cells, pulsed with or without RMF or RHAMM-3 peptides initially wereused to determine the binding specificity of the mAb. Three out of fivehuman IgG1, including WT1 ab1, showed specific binding to the T2 cellsthat were pulsed only with WT1 peptide, but not to T2 alone or T2 pulsedwith control peptide RHAMM-R3. The binding avidity of the mAb weresubstantially enhanced (50 to 100 fold), compared to their parental scFvphage clones. Two mAbs among the five showed binding to T2 cells aloneor pulsed with the control peptide RHAMM-R3, although the binding wasgreatly enhanced by pulsing the cells with RMF peptide. This suggestedthat these two mAb also had high avidity for epitopes on the HLA-A2molecule alone and therefore were excluded from further investigation.This was not unexpected, as it has been a common problem for producingsuch mAb against peptide/MHC complexes, given the predominance of theMHC class I molecule epitopes within the complexes. It also suggeststhat the precise specificity of the mAb for the complexes might not bedetermined easily at the scFv stage, due to the lower affinity comparedto the bivalent IgG1 mAb.

The binding affinity of the three remaining mAb specific for the WT1p/A2complex first was investigated on T2 cells pulsed with or without RMFand control RHAMM-R3 peptides (50 ug/ml) by titration of the mAbs. MabWT1 ab1 showed the strongest binding, down to a concentration of 0.01ug/ml. Isotype control human IgG1 showed no binding at anyconcentrations tested (FIG. 5). In addition to WT1 ab1, the two othermAb, WT1 ab3 and WT1 ab5, showed specific binding at a range of <1 ug/mlof the mAb concentrations used. The specific recognition of the mAb alsodepended on the antigenic density on the cell surface. T2 cells werepulsed with RMF or R3 peptides at 50, 25, 12.5, 6.25, 3.13 and 1.6ug/ml; the test mAb were used at 1 ug/ml for the T2 binding assay. WT1ab1 could detect the RMF peptide/A2 complex on T2 cells in aconcentration-dependent manner at concentrations as low as 1.6 ug/ml,with significantly higher fluorescence intensity than the other 2 mAb(FIG. 6). These results further confirmed that the WT1 ab1 possessed thehighest avidity for the RMFp/A0201 complex.

Example 4

Epitope Mapping.

To investigate with more precision the epitope for WT1 ab1 recognition,RMF peptides were substituted at positions 1, 3, 4, 5, 6, 7 and 8 withalanine and pulsed onto T2 cells and were tested for binding of WT1 ab1.Positions 2 and 9 of the RMF were left intact, as these are the anchorresidues for peptide binding to the HLA-A0201 molecule. Except forposition 1, alanine substitutions at other positions did not greatlyaffect the binding of the WT1 ab1, as compared to the native RMF peptide(FIG. 19). However, substitution of position 1 by either alanine(WT1-A1-B) or tyrosine (WT1-A1), completely abrogated the binding of WT1ab1. The loss of binding was not due to the reduction of peptide bindingaffinity to the HLA-A2 molecule, as both peptides showed the strongestbinding in T2 stabilization assay using the mAb specific for the HLA-A2molecule, clone BB7 (FIG. 20). These results show that the arginine atposition 1 of the RMF peptide is one of the most crucial for the WT1 ab1recognition. The role of the residues at positions #2 and 9, could notbe assessed.

The next important question was whether WT1 ab1 was able to recognizenaturally processed WT1 epitope RMF presented by HLA-A0201 molecules onthe cell surface. A panel of cell lines was selected based on theexpression of WT1 mRNA and HLA genotyping (Table 12).

TABLE 12 Ratio of HLA-A2 WT1 WT1 AB BB7.2: genotype mRNA binding IsotypeMesothelioma/solid tumor JMN + + + 248 Meso 37 + + + 68 Meso 47 +(02xx) + + 17 H2452 + + + 20 Meso34 + + + 37.3 Meso-56 + (02xx) + + 23H2373 + + − 1.6 MSTO − + − 1.4 VAMT − 3+ − NT Mewo + − − 3 Leukemias andother hematopoietic cell lines BV173 + ++ + 196 BA25 + ? + 117.5ALL-3 + + + 60 U266 + + − 1.8 697 + 5+ − 4.1 LAMA + 2+ − 6 SKLY-16 + − −1.9 HL-60 − 3+ − 0.4 K562 − 2+ − 1.5 T2 + NT − >20

WT1 mRNA expression level was estimated according to a previous study(Rena), by quantitative RT-PCR.

Among 7 human mesothelioma cell lines that are positive for bothHLA-A0201 and WT1 mRNA, WT1 ab1 bound to 6 out of 7 cell lines, but notto the cells that were either HLA-A0201 negative (MSTO and VAMT) or WT1mRNA negative, such as melanoma cell line, Mewo (FIG. 21).

Similarly, among 9 leukemia cell lines tested, WT1 ab1 bound to 3 celllines BV173 (FIG. 22), BA25 and ALL-3, that are positive for both WT1mRNA and HLA-A0201, but not to HLA-A2-negative cell lines HL60 and K562,that have been demonstrated to express a high level of WT1 transcriptsin numerous studies.

As expected, intensity of binding of the WT1 AB1 also appeared to bedirectly associated with the expression level of HLA-A0201 molecule, asshown in mesothelioma cells H2373, leukemia cell lines 697 and LAMA, andmyeloma cell line U266. Although these cell lines were positive for bothWT1 transcripts and HLA-A2, the expression level of the HLA-A2 was low(Table 12) and the mAb did not show binding. On the other hand, theresults obtained with T2 cells argue against the possibility of WT1 ab1binding to HLA-A0201 alone as T2 cells expressed a high level of HLA-A2molecule. Notably, WT1 ab1 did not bind to T2 cells alone or pulsed withR3 and other HLA-A0201-binding peptides such as Ewing sarcoma-derived(EW) or the heteroclitic peptide for the RMF peptide, WT1-A1; these twopeptides have been shown to have higher affinity for the HLA-A0201molecule in T2 stabilization assay (28). These results provided strongevidences that WT1 ab1 recognition was specific for epitopes jointlycomposed of the RMF peptide and the A0201 molecule in a complex. Thebinding of the other two mAb, WT1 ab3 and WT1 ab5, to the BV173 and JMNcells was also weaker than WT1 ab1.

Example 5

Quantitation of WT1 ab1 Binding Sites on Cells.

A radioimmunoassay using 1251-labeled WT1 ab1 was used to confirm thespecificity of the antibody for WT1⁺ HLA-A0201⁺ cell lines, to determinean affinity constant and to assess the number of antibody binding sitesper cell on a panel of cell lines. Scatchard analysis based on bindingto JMN cells showed an avidity constant of about 0.2 nM (FIG. 23). Thisnumber was confirmed by interferometry using a Forte Bio device.125-I-labeled WT1 ab1 was used to confirm the specificity of theantibody for WT1⁺ HLA-A0201⁺ cell lines, and to assess the number ofantibody binding sites on a panel of cell lines (FIG. 24). Because wecannot determine whether the bivalent mAb is binding to 1 or 2 complexeson the surface, total epitopes per cell could be as high as twice thenumber of mAb binding sites. Again, WT1 ab1 bound to JMN, ALL-3, BA25,BV173, which are positive for both HLA-A0201 and WT1 mRNA, but notHLA-A0201 negative (HL60) or WT1 mRNA negative (SKLY-16) cells. WT1 ab1did not bind to 697 cells, which are both HLA-A0201 and WT1 positive,but contain low levels of HLA-A0201 (Table 12), confirming that acertain level of total MHC complex is needed to present sufficient WT1peptide for WT1 ab1 binding. T2 pulsed with RMF bound the highest numberof mAb (50,000 per cell), followed by JMN cells which bound ˜6×10³ WT1ab1 molecules per cell, translating to between 6×10³ and 1.2×10⁴RMFpeptide/A2 complexes per cell assuming monovalent or bivalentantibody binding, respectively. The three positive leukemia cell linesbound between 1×10³ and 2×10³ WT1 ab1 molecules, or 2×10³-4×10³ bindingsites (FIG. 24). These results were confirmed by quantitative flowcytometry.

Example 6

WT1 Ab 1 Binding to Leukemic Patient Samples.

We next investigated if WT1 ab1 is able to detect the RMF epitope onprimary AML cells. Radioimmunoassay showed significant binding of theWT1 AB1 to AML blasts of patient 1, who is HLA-A2 positive and WT1mRNA⁺. WT1 ab1 bound to CD33⁺ and CD34⁺ double positive cells thataccount for more than 83% of the whole cell populations (FIG. 25). WT1ab1 did not bind to the cells of 3 other patients who are either HLA-A2positive but mRNA negative or HLA-A2 negative. WT1 ab1 did not bind toPBMCs from either HLA-A2 positive or negative healthy donors. Theresults were confirmed by flow cytometry analysis. WT1 AB1 did not showsignificant binding to the blasts from the patients who were A0201negative (FIG. 26). The results were consistent with the resultsobtained with mRNA expression of the cells. These data confirm that thelevel of RMFp/HLA-A0201 on the surface of leukemia cells is adequate toallow reactivity with the WT1 ab1 and the levels on WT1 negative healthycells is not significant.

Example 7

WT1 AB1 Mediates ADCC Against Tumor Cells

ADCC is considered to be one of the major effector mechanisms oftherapeutic mAb in humans. In the presence of human PBMC, WT1 ab1mediated dose-dependent PBMC ADCC against the T2 cells loaded with RMFpeptide, but not T2 cells alone or T2 cells pulsed with control R3peptide (FIG. 27). Importantly, WT1 ab1 was able to mediate ADCC againstnaturally presented RMF epitope by HLA-A0201 molecule on tumor cells,such as the mesothelioma cell line, JMN (FIG. 33), and the leukemia cellline BV173 (FIG. 34), but not the HLA-A2 negative cells MSTO (FIG. 28)or HL-60 (FIG. 29). The killing was consistently observed at 1 μg/ml orbelow of WT1 ab1 using PBMCs as effector cells from multiple healthydonors. Importantly, WT1 ab1 also killed primary A0201-positive AMLblasts that were positive for the WT1 ab1 binding, but not the blaststhat were HLA-A0201 negative (FIG. 30). These results demonstrated thatWT1 ab1 mediates specific ADCC against cells that naturally express RMFand HLA-A0201 complex at physiologic levels as well as on cell lines.

Example 8

WT1 AB1 Eliminates Human Leukemia Cells in NSG Mice

The efficacy of WT1 ab1 in vivo was tested in NOD SCID gamma (NSG) micexenografted intravenously 6 days previously with BV173 bcr/abl positiveacute lymphoblastic leukemia. At the time of treatment, mice hadleukemia in their liver, spleen, and BM visible by luciferase imaging.NSG mice lack mature B-, T- and NK-cells, and we hypothesized thatintroducing human effector cells (CD3⁻, CD34⁻, PBMCs) along with WT1 ab1treatment would recapitulate in vivo the ADCC-mediated anti-tumoreffects observed in vitro. Injection of effectors along with two 100 μgdoses of WT1 ab1 nearly ablated tumor growth compared to controls (FIG.31). This effect was durable over the course of the experiment (FIG.32). Interestingly, early on in the trials, effector cells alone orcombined with control IgG appeared to promote more rapid growth ofleukemia relative to mice injected with leukemia alone, demonstratingthat the anti-tumor effect was unrelated to the effectors by themselves.Several of the mice given effectors (with or without control mAb) diedearly in the experiment with massive infiltration of the BV173.

Surprisingly, WT1 ab1 treatment without human effectors alsodramatically reduced tumor burden as well as the WT1 ab1 combined witheffectors for approximately 30 days (FIG. 32), though tumors eventuallyrelapsed far more quickly in the WT1 ab1 alone group, when compared toWT1 ab1 combined with effectors group (FIG. 32). We confirmed the effectof WT1 ab1 alone and titrated the dosage to evaluate potency. WT1 ab1alone produced a marked reduction in tumor burden at early time pointsat all doses tested (25-100 μg times 2 doses). Tumors in all treatmentgroups relapsed slowly after antibody therapy was stopped; and by day 23(13 days after the last antibody injection), significantly more tumorrelapse could be observed in the 25 μg group compared to the 100 μg dosegroup, indicating a dose-response to WT1 ab1 therapy (FIG. 33). Beforetreatment, mice displayed the largest tumor burden in the liver, whichwas quickly cleared by WT1 ab1. Upon relapse, tumor in the highest dosegroup appeared to develop mainly in bone marrow, while tumor returnedmore quickly to the liver in mice treated with the lowest dose.

Example 9

Engineering Antibodies to Enhance their Cytotoxic Abilities.

Bispecific antibodies are constructed that recognize both WT1/A2 complexand CD3 on immune T cells as described (43,44) with a human IgG1 Fc.Bispecific antibodies are expected to recruit and target cytotoxic Tcells to WT1/A2 positive cancer cells, while maintaining Fc effectorfunctions and long half life in vivo. Three mechanisms are involved inthe specific killing of cancer cells mediated by bispecific antibodies:i) killing by activated T cells; ii) ADCC activity; iii) CDC activity.Other formats of bispecific antibodies can be constructed, such tandemscFv molecules (taFv), diabodies (Db), or single chain diabodies (scDb),and fusion protein with human serum albumin (45, 46, 47, 48), but aredevoid of Fc effector functions with distinct pharmacokinetic profiles.

WT1/A2 target specific-ADCC activity is enhanced by expressingantibodies recombinantly in glycol-engineered CHO cells as described inU.S. Pat. Nos. 8,025,879; 8,080,415; and 8,084,022. The modifiedoligosaccharide N-glycan on Asn297 alters effector functions asfollows: 1) higher affinity binding to CD16/FcRIIIa for improved ADCCactivity mediated by human Natural Killer cells; 2) reduced bindingaffinity to CD32b/FcRIIb, an inhibitory receptor expressed in multipletypes of immune cells (except NK cells), for improved ADCC activitymediated by effector cells such as neutrophils and antigen presentationby macrophage and DC cells (50, 51, 52). Enhanced antibodies areexpected to achieve better efficacy for cancer treatment in vivo.

Glycosylation (specifically fucosylation) variants of IgG Fc can beproduced using host expression cells and methods described in U.S. Pat.Nos. 8,025,879; 8,080,415; and 8,084,022, the contents of which areincorporated by reference. Briefly, messenger RNA (mRNA) coding forheavy or light chain of the antibodies disclosed herein, is obtained byemploying standard techniques of RNA isolation purification andoptionally size based isolation. cDNAs corresponding to mRNAs coding forheavy or light chain are then produced and isolated using techniquesknown in the art, such as cDNA library construction, phage libraryconstruction and screening or RT-PCR using specific relevant primers. Insome embodiments, the cDNA sequence may be one that is wholly orpartially manufactured using known in vitro DNA manipulation techniquesto produce a specific desired cDNA. The cDNA sequence can then bepositioned in a vector which contains a promoter in reading frame withthe gene and compatible with the low fucose-modified host cell.

Numerous plasmids that contain appropriate promoters, control sequences,ribosome binding sites, and transcription termination sites, andoptionally convenient markers are known in the art, these include butare not limited to, vectors described in U.S. Pat. Nos. 4,663,283 and4,456,748. In one embodiment, the cDNA coding for the light chain andthat coding for the heavy chain may be inserted into separate expressionplasmids. In an alternative embodiment, the cDNA coding for the lightchain and that coding for the heavy chain may be inserted together inthe same plasmid, so long as each is under suitable promoter andtranslation control. Results are shown in FIG. 34.

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What is claimed is:
 1. A bispecific antibody comprising: a first antigenbinding fragment, comprising one of: (a) a heavy chain (HC) variableregion comprising HC-CDR1, HC-CDR2 and HC-CDR3 respectively, comprisingamino acid sequences SEQ ID NOS: 2, 3, and 4; and a light chain (LC)variable region comprising LC-CDR1, LC-CDR2 and LC-CDR3 respectively,comprising amino acid sequences SEQ ID NOS: 8, 9 and 10; (b) a heavychain (HC) variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3respectively, comprising amino acid sequences SEQ ID NOS: 20, 21 and 22;and a light chain (LC) variable region comprising LC-CDR1, LC-CDR2 andLC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 26, 27and 28; (c) a heavy chain (HC) variable region comprising HC-CDR1,HC-CDR2 and HC-CDR3 respectively, comprising amino acid sequences SEQ IDNOS: 38, 39 and 40; and a light chain (LC) variable region comprisingLC-CDR1, LC-CDR2 and LC-CDR3 respectively, comprising amino acidsequences selected from SEQ ID NOS: 44, 45 and 46; (d) a heavy chain(HC) variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3respectively, comprising amino acid sequences SEQ ID NOS: 56, 57 and 58;and a light chain (LC) variable region comprising LC-CDR1, LC-CDR2 andLC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 62, 63and 64; (e) a heavy chain (HC) variable region comprising HC-CDR1,HC-CDR2 and HC-CDR3 respectively, comprising amino acid sequences SEQ IDNOS: 74, 75 and 76; and a light chain (LC) variable region comprisingLC-CDR1, LC-CDR2 and LC-CDR3 respectively, comprising amino acidsequences SEQ ID NOS: 80, 81 and 82; or (f) a heavy chain (HC) variableregion comprising HC-CDR1, HC-CDR2 and HC-CDR3 respectively, comprisingamino acid sequences SEQ ID NOS: 92, 93 and 94; and a light chain (LC)variable region comprising LC-CDR1, LC-CDR2 and LC-CDR3 respectively,comprising amino acid sequences SEQ ID NOS: 98, 99 and 100; and a secondantigen binding fragment that binds to CD3.
 2. The bispecific antibodyof claim 1, wherein the first antigen binding fragment, comprises aV_(H) and V_(L) comprising first and second amino acid sequences,respectively, selected from SEQ ID NOS: 14 and 16; 32 and 34; 50 and 52;68 and 70; 86 and 88; and 104 and
 106. 3. The bispecific antibody ofclaim 1, wherein the first antigen binding fragment, comprises an aminoacid sequence selected from SEQ ID NOS: 18, 36, 54, 72, 90, and
 108. 4.The bispecific antibody of claim 1, wherein first antigen bindingfragment specifically binds to an WT1 peptide bound to HLA-A2.
 5. Thebispecific antibody of claim 4, wherein said WT1 peptide has the aminoacid sequence RMFPNAPYL (SEQ ID NO: 1).
 6. The bispecific antibody ofclaim 4, wherein said HLA-A2 is HLA-A0201.
 7. A method for treatment ofa subject having a WT1-positive disease, comprising administering to thesubject a therapeutically effective amount of the bispecific antibody ofclaim
 1. 8. The method of claim 7, wherein the WT1-positive disease is achronic leukemia or acute leukemia or WT1⁺ cancer.
 9. The method ofclaim 8, wherein the WT1-positive disease is selected from the groupconsisting of chronic myelocytic leukemia, multiple myeloma (MM), acutelymphoblastic leukemia (ALL), acute myeloid/myelogenous leukemia (AML),myelodysplastic syndrome (MDS), mesothelioma, ovarian cancer,gastrointestinal cancers, breast cancer, prostate cancer andglioblastoma.
 10. A pharmaceutical composition comprising the bispecificantibody of claim 1.